A seat belt retractor including a torsion bar is provided. A second torque transmitting portion of the torsion bar is clamped between a reel and a holder member press-fitted radially in the reel without backlash in the axial direction. A first torque transmitting portion is strictly fixed to a pawl holder without backlash in the axial direction by radially crimping an internal periphery of a left end portion of the pawl holder of a locking means. Therefore, the first and second torque transmitting portions conventionally formed in a torsion bar are effectively used, thereby eliminating a groove for an E-ring as conventionally used and thus simplifying the configuration of the torsion bar. In addition, since the first and second torque transmitting portions can operate without backlash, the occurrence of jarring noise due to vibrations of the seat belt retractor can be prevented.

The present invention relates to a seat belt retractor for a webbing that permits the webbing to be retracted and withdrawn. More particularly, the present invention relates to a seat belt retractor with a torsion bar for absorbing load exerted on a webbing when an occupant puts on the webbing and the webbing is stopped from being withdrawn in the event of emergency such as a vehicle collision at which large deceleration is exerted on the vehicle.
Conventionally, a seat belt device installed in a vehicle such as an automobile prevents an occupant from being thrown out of a vehicle seat by restraining the occupant with a webbing. The seat belt protects the occupant in the event of emergency such as mentioned above.
A typical seat belt device is provided with a seat belt retractor for accommodating a webbing. When not used, the webbing is fully wound on a reel of the retractor. When an occupant puts on the webbing, the seat belt is withdrawn to extend across the occupant. The seat belt retractor includes a locking mechanism that is activated in the event of emergency as mentioned above to stop the rotation of the reel in an unwinding direction, thereby preventing the webbing from being withdrawn. Therefore, the seat belt webbing can securely restrain and protect the occupant.
However, conventional seat belt retractors being used in seat belt devices have a drawback or problem. That is, when the webbing restrains and protects the occupant, a large deceleration is exerted on the vehicle so that the occupant tends to move forward due to a large inertia force. Accordingly, a large load is exerted on the webbing and the occupant receives a large impact load from the webbing. Thus, there remains a need to absorb the impact load to reduce the discomfort for the occupant. Though this impact load is not so severe for the occupant, it is better to absorb the impact load.
FIG. 8 is a vertical sectional view showing an example of a conventional seat belt retractor 1 including a torsion bar. As shown in the drawing, the seat belt retractor, includes a U-like frame 2, a webbing 3, a reel 4 which is rotatably supported between side walls of the U-like frame 2 and on which the webbing 3 is wound, a deceleration sensing means 5 which detects a large deceleration of the vehicle occurred in case of emergency mentioned above, a locking mechanism 6 which is activated by the deceleration sensing means 5 to prevent the reel from rotating in the webbing unwinding direction, a torsion bar 7 loosely fitted and inserted in the axial direction in the center of the reel 4 and which rotatably connects the reel 4 and the locking mechanism 6, a spring means 8 for always biasing the reel 4 in the webbing winding direction through a bush 10 by the spring force of a spiral spring 9, a pretensioner 11 which is activated in the case of emergency mentioned above for producing webbing winding torque, and a bush 12 for transmitting the webbing winding torque of the pretensioner 11 to the reel 4.
The locking mechanism 6 include a pawl holder 14 which rotates together with a first torque transmitting portion 17 of the torsion bar 7. The holder 14 pivotally holds the pawl 13 and also includes a lock gear 6a which rotates together with the torsion bar 7 during normal operation. The lock gear 6a stops in an emergency corresponding to the activation of the deceleration sensing means 5 to produce rotation relative to the torsion bar 7 and engages the pawl 13 with one of internal teeth 19 of the side wall of the frame 2, thereby stopping the rotation of the pawl holder 14 and the rotation of the reel 4 in the webbing unwinding direction. The pawl holder 14 has an external thread portion 15 that includes a nut-like stopper member 16 which rotates together with the reel 4 and is screwed into the thread portion 15.
The torsion bar 7 includes the first torque transmitting portion 17 which is fitted to the pawl holder 14 not allowing relative rotation therebetween, and a second torque transmitting portion 18 which is fitted to the reel 4 not allowing relative rotation therebetween.
The reel 4 is always biased by the spring force of the spring means 8 in the webbing winding direction through the bush 10, the torsion bar 7, the second torque transmitting portion 18 of the torsion bar 7, and the bush 12. The webbing winding torque produced by the pretensioner 11 is transmitted to the reel 4 through the bush 12 whereby the reel 4 winds up the webbing 3 a predetermined amount.
In the conventional seat belt retractor 1 having the aforementioned structure, the webbing 3 is fully wound up by the biasing force of the spring means 8 when not being used by a vehicle passenger. As the webbing 3 is withdrawn at a normal speed for putting on, the reel 4 rotates in the webbing unwinding direction so that the webbing 3 is smoothly withdrawn. After a tongue (not shown) slidably fitted to the webbing 3 is inserted into and engaged with a buckle fixed to a vehicle body, the excess amount of the webbing 3 is wound back onto the reel 4 by the biasing force of the spring means 8 until the webbing 3 is fitted to the occupant""s body without excess stress.
In an emergency, such as mentioned above, the webbing winding torque produced by the pretensioner 11 is transmitted to the reel 4 so that the reel 4 winds up the webbing 3 for a predetermined amount, in order to strongly restrain the occupant. The deceleration sensing means 5 is activated by a large deceleration produced by the emergency condition to activate the locking mechanism 6. The activation of the deceleration sensing means 5 prevents the rotation of the lock gear 6a in the webbing unwinding direction. In addition, the pawl 13 of the locking mechanism 6 pivots and engages one of the internal teeth 19 of the side wall of the frame 2 so as to stop the rotation of the pawl holder 14. As a result of this, the reel 4 rotates in the webbing unwinding direction relative to the pawl holder 14 while the torsion bar 7 is twisted. By this twisting of the torsion bar 7, the load applied to the webbing 3 is limited, thus absorbing the impact on the occupant.
Since the reel 4 rotates relative to the paw holder 14, the stopper member 16 rotates relative to the external thread portion 15 which is screwed into the stopper member 16, whereby the stopper member 16 moves toward the pawl holder 14. When stopper member 16 comes into contact with the pawl holder 14, the stopper member 16 is prevented from further rotation. As a result, the reel 4 is also prevented from rotating and the torsion bar 7 is prevented from being twisted. In this manner, the webbing 3 is stopped from being withdrawn whereby the occupant is restrained by the webbing 3. Thus, the movement of the stopper member 16 defines the maximum allowable twisting deformation of the torsion bar 7. Through adjustment the EA (xe2x80x9cEnergy Absorbingxe2x80x9d) stroke as a rotatable stroke of the reel 4 relative to the pawl holder 14 can be freely set.
In addition, in the conventional seat belt retractor 1, the pawl holder 14 of the locking mechanism 6 rotates in the webbing unwinding direction relative to the lock gear 6a when the webbing is rapidly withdrawn. Also in this case, the pawl 13 of the locking mechanism 6 engages one of the internal teeth 19 of the side wall of the frame 2 so as to stop the rotation of the pawl holder 14 in the same manner as mentioned above. Therefore, the reel 4 is prevented from rotating in the unwinding direction through the torsion bar 7, thus stopping the withdrawal of the webbing.
In the conventional seat belt retractor 1, the torsion bar 7 extends to penetrate the bush 12. The bush 12 is sandwiched between the second torque transmitting portion 18 of the torsion bar 7 and an E-ring 20 fitted to a groove of the torsion bar 7 whereby the movement of the torsion bar 7 in the axial direction relative to the bush 12 is limited. The relative movement of the bush 12 in the axial direction relative to the frame 2 is limited. Thus, the movement of the torsion bar 7 relative to the frame 2 is limited. The torsion bar 7 is fixed in the axial direction relative to the frame 2 and the reel 4.
In the conventional seat belt retractor 1, the groove in which the E-ring 20 is fitted is required to be formed in the torsion bar 7. This arrangement complicates the configuration of the torsion bar 7. In addition, since the torsion bar 7 extends to penetrate the bush 12, the torsion bar 7 should have a long axial length.
Furthermore, there can be backlash due to the dimensional tolerance between the E-ring 20 and the groove in which the E-ring 20 is fitted. The backlash may cause a jarring noise when the vehicle vibrates for running. A mechanism must be provided to reduce the jarring noise. Thus, the structure for fixing the torsion bar 7 in the axial direction is complex thereby reducing the workability of assembling the torsion bar 7, and increasing the cost.
Thus, there remains a need for a seat belt retractor which can be manufactured at a low cost and which can realize the simplification of the configuration of a torsion bar, the simplification of the structure for fixing the torsion bar in the axial direction, and the improvement of the workability of assembling the torsion bar, and can prevent the occurrence of a jarring noise.
To solve the aforementioned problems, an improved seat belt retractor is provided. The seat belt retractor comprises a reel for winding up a webbing; a locking means for preventing the rotation of the reel in the webbing unwinding direction when actuated; a deceleration sensing means which detects a predetermined deceleration to actuate the locking means; and a torsion bar rotationally connecting the reel and the locking mechanism. The torsion bar having a portion disposed within the reel coaxially with the reel, the torsion bar absorbing load exerted on the webbing when the locking means is actuated. The torsion bar comprises a flange-like first torque transmitting portion which is fitted to the locking means so that the first torque transmitting portion is connected to the locking means to rotate together. The torsion bar also includes a second torque transmitting portion which is fitted to the reel so that the second torque transmitting portion is connected to the reel to rotate together. The torsion bar further includes a torsion portion which is fixed between the first and second torque transmitting portions and is designed to be twisted to absorb the load exerted on the webbing. One end in the axial direction of at least one of the first and second torque transmitting portions is axially in contact with the corresponding one of the locking means. The reel and the other end of the at least one of the torque transmitting portions is axially in contact with a holder member fitted in the corresponding one of the locking means and the reel.
According to an alternative embodiment of the present invention, a seat belt retractor is provided. The seat belt retractor comprises a reel for winding up a webbing; a locking means for preventing the rotation of the reel in the webbing unwinding direction when actuated; a deceleration sensing means which detects a predetermined deceleration to actuate the locking means; and a torsion bar rotationally connecting the reel and the locking mechanism. The torsion bar having a portion disposed within the reel coaxially with the reel. The torsion bar absorbing load exerted on the webbing when the locking means is actuated. The torsion bar comprises a flange-like first torque transmitting portion which is fitted to the locking means so that the first torque transmitting portion is connected to the locking means to rotate together. The torsion bar also includes a second torque transmitting portion which is fitted to the reel so that the second torque transmitting portion is connected to the reel to rotate together. The torsion bar further includes a torsion portion which is fixed between the first and second torque transmitting portions and is designed to be twisted to absorb the load exerted on the webbing. One end in the axial direction of at least one of the first and second torque transmitting portions is axially in contact with the corresponding one of the locking means. The reel and the other end of the at least one of the torque transmitting portions is axially in contact with a crimped portion of the corresponding one of the locking means and the reel.
According to another alternative embodiment of the present invention, a seat belt retractor is provided. The seat belt retractor comprises a reel for winding up a webbing; a locking means for preventing the rotation of the reel in the webbing unwinding direction when actuated; a deceleration sensing means which detects a predetermined deceleration to actuate the locking means; and a torsion bar rotationally connecting the reel and the locking mechanism. The torsion bar having a portion disposed within the reel coaxially with the reel. The torsion bar absorbing load exerted on the webbing when the locking means is actuated. The torsion bar comprises a flange-like first torque transmitting portion which is fitted to the locking means so that the first torque transmitting portion is connected to the locking means to rotate together. The torsion bar also includes a second torque transmitting portion which is fitted to the reel so that the second torque transmitting portion is connected to the reel to rotate together. The torsion bar further includes a torsion portion which is fixed between the first and second torque transmitting portions and is designed to be twisted to absorb the load exerted on the webbing. One end in the axial direction of one of the first and second torque transmitting portions is axially in contact with the corresponding one of the locking means. The reel and the other end of the one of the torque transmitting portions is axially in contact with a holder member fitted in the corresponding one of the locking means and the reel. One end in the axial direction of the other one of the first and second torque transmitting portions is in axially in contact with the other corresponding one of the locking means. The reel and the other end of the other one of the torque transmitting portions is axially in contact with a crimped portion of the other corresponding one of the locking means and the reel.
Furthermore, the holder member may be composed of a pin having a slope(s) where it comes in contact with the torque transmitting portion.
Furthermore, the holder member may further includes a slit(s) formed in the portion where comes in contact with the torque transmitting portion so as to elastically press the torque transmitting portion in the axial direction.
Furthermore, the holder member may be composed of a plate to elastically press the torque transmitting portion in the axial direction.
In addition, the holder member may be composed of a pin having a first engaging protrusion at one end thereof and a first engaging protrusion at the other end thereof. A member into which the holder member is fitted is formed with a holder-member-fitting hole having two stepped portions. The first engaging protrusion is engaged with one of the stepped portions and the second engaging protrusion is engaged with the other one of the stepped portions when the holder member is inserted into the holder-member-fitting hole. As a result, the torque transmitting portion is brought in contact with a middle portion of the pin.
Moreover, at least one of the first and second torque transmitting portions may be formed in serration, to have a TORX-wrench-like section, or to have a hexagonal section.
At least one of the first and second torque transmitting portions may be clamped between one of the locking means and the reel, into which the at least one of torque transmitting portions is fitted, and the holder member in the axial direction. Alternatively, at least one of the first and second torque transmitting portions is fixed by crimping one of the locking means and the reel into which the at least one of torque transmitting portions is fitted. Alternatively, one of the first and second torque transmitting portions may be clamped between one of the locking means and the reel, into which the at least one of torque transmitting portions is fitted, and the holder member in the axial direction. The other one of the first and second torque transmitting portions may be fixed by crimping one of the locking means and the reel into which the at least one of torque transmitting portions is fitted.
Accordingly, at least one of the first and second torque transmitting portions conventionally formed in the torsion bar is effectively used. Therefore, a groove into which an E-ring conventionally used for the torsion bar can be eliminated, thereby simplifying the configuration of the torsion bar and simplifying the working for the torsion bar.
At least one of the first and second torque transmitting portions may be strictly fixed without backlash in the axial direction. Therefore, the occurrence of jarring noise due to vibrations or the like of the seat belt retractor can be prevented. The torque transmission can be further secured.
As apparent from the above description, at least one of first and second torque transmitting portions conventionally formed in a torsion bar is effectively used to fix the torsion bar in the axial direction. An E-ring as conventionally used for the torsion bar can be eliminated, thereby simplifying the configuration of the torsion bar. Therefore, the working for assembling the torsion bar is also simplified and the manufacturing cost of the torsion bar can be reduced.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention as claimed.


A pressure control apparatus is used in an apparatus for effecting extraction, chromatographic separation, and fractionation. The pressure control apparatus is intended to reduce the amount of a fluid stagnating in a valve, and is arranged to cyclically open and close the valve, thereby controlling the level of pressure. An apparatus for effecting extraction, chromatographic separation, and fractionation is designed to perform in an on-line manner the extraction of soluble components contained in a sample as well as the separation and fractionation of the components. A chromatography is connected to a downstream side of a supercritical fluid extraction device, and the aforesaid pressure control apparatus is used as back-pressure regulators (9, 18) for the chromatography and the extraction device. An extraction fluid and an elution fluid which are used in a mobile phase may be a common fluid (FIG. 1) or separate fluids (FIG. 10). At the time of extraction, the soluble components of the sample within an extraction vessel (6) is extracted by a supercritical fluid, and is delivered to an adsorption column (14). The supercritical fluid is converted into a gas by reducing the pressure within the adsorption column and thus the solubility of the fluid is lowered so that the resultant extract is adsorbed by an adsorbent packed in this column. At the time of chromatographic separation, the pressure within the adsorption column (14) is maintained at a critical pressure or higher and thus an extract is eluted by the supercritical fluid and is delivered to a separation column (16), in which the extract is separated into individual components. Then, when the pressure within the adsorption column is reduced, the supercritical fluid is gasified and the result components are collected as fractions.

1. Field of the Invention
The present invention relates to a pressure control apparatus for providing control over the fluid pressure of a gas or a liquid within a container or a flow passage, and to an apparatus employing the same, that is, an apparatus for effecting extraction, chromatographic separation and, fractionation which apparatus is arranged to extract a soluble substance from a sample by the use of a supercritical fluid or a liquefied gas, introduce the resultant extract into a chromatograph in an on-line manner for separation into individual components, and collect them as fractions.
2. Description of the Prior Art
The present inventor proposed an extraction/chromatographic separation apparatus capable of extracting a soluble substance from a sample by using a supercritical fluid or a liquefied gas and introducing the resultant extract into a chromatograph in an on-line manner, thereby separating the individual components of that extract (Journal of Chromatography, 332 (1985), pages 107 to 116).
The aforementioned extraction/chromatographic separation apparatus is arranged to deliver an extraction/elution fluid containing the supercritical fluid or the liquefied gas by means of a pump to the interior of an extraction vessel including a sample, extract a soluble component from the sample in the extraction vessel, cause the extract primarily obtained in a flow passage downstream of the extraction vessel to be drawn at a low pressure into a trapping loop previously charged with the extraction/elution fluid in the gaseous phase thereof, temporarily store that extract in that trapping loop, and introduce the extract to the chromatograph in an on-line manner.
A back pressure control valve for allowing the extract to be separated into its individual components in a separation column and for introducing the resultant components into a detector is disposed downstream of the detector which constitutes a part of the chromatograph.
The above-described apparatus accomplishes a substantial improvement in that extraction and separation can be effected in an on-line manner.
However, it is necessary to set the trapping loop apart from the extraction vessel and the separation column of the chromatograph, to connect the trapping loop to only one of the extraction vessel and the separation column, or to connect the trapping loop directly to the pump. Therefore, three six-way valves are needed, and this necessity disadvantageously complicates the construction and operation of the apparatus.
In general, typical prior art pressure control apparatus have been arranged to provide control so that a detection pressure may be set to a desired pressure by adjustment of spring force which is loaded on the pressure sensing piston, on which a value needle is installed so as to change the opening of a pressure control valve. Such a valve is commercially available from Tescom (MN).
However, since this pressure control valve is arranged to adjust the opening of the gap between a valve seat and a needle member by the movement of the pressure sensing piston, the volumes of the spaces defined upstream and downstream of that gap are relatively large and therefore the amount of fluid stagnating in the valve may reach at least several to several tens of milliliters.
As a result, even if separation is complete in the separation column, for example, in the previously-mentioned supercritical fluid chromatograph, an effluent may flow from the column into the pressure control valve to cause re-mixture of the separated components.
To the end of preventing such re-mixture, in a supercritical fluid chromatograph of the type employing a capillary column, a capillary tube having an inner diameter of 10 m or less is connected to a downstream side of the separation column so that the flow resistance of the supercritical fluid is utilized to maintain a required level of fluid pressure (Journal of Chromatographic Science, Vol. June 14, 1986, pages 236 to 241).
However, in order to vary the level of pressure, the flow rate of the fluid in the mobile phase must also be varied, with the result that the pressure and the flow rate both of which determine the period of retention time are varied at the same time. Therefore, it has been impossible to examine the details of the characteristics of the retention time by independently varying only one of the pressure and the flow rate.
It is known that a method of effecting separation under reduced pressure is widely used in supercritical fluid extraction apparatus. In this method, the level of pressure is lowered to cause a reduction in the solubility of an extract in the supercritical fluid, thereby separating and collecting a desired solute from the fluid.
However, if the aforesaid pressure control valve arranged to adjust the valve opening is used in a small supercritical fluid extraction apparatus, the following problems are encountered. Since a large amount of fluid stagnates in the pressure control valve, individual separated extracts are re-mixed within the pressure control valve, and this makes it impossible to fractionate the individual extracted substances. In addition, since the extracts stagnate in the valve when the pressure of the fluid, i.e., extraction medium, decreases rapidly causing solubility reduction, they are easily precipitated and adhered onto inner walls of the valve, thereby hindering the extracted substances from being completely fractionated. In addition, such precipitated substances often block the flow path of the valve.

Pressure control apparatus and apparatus for effecting extraction chromatographic separation, and fractionation by employing the same


Methods are disclosed for screening for the occurrence of gene silencing (e.g., post transcriptional gene silencing) in an organism. Also provided are methods for isolating silencing agents so identified.

The present invention relates generally to methods and materials for use in achieving and detecting gene silencing, particularly post-transcriptional gene silencing, in an organism.
Methods of detecting and efficiently achieving gene silencing are of great interest to those skilled in the art.
Post-transcriptional gene silencing (PTGS) is a nucleotide sequence-specific defence mechanism that can target both cellular and viral mRNAs. PTGS occurs in plants and fungi transformed with foreign or endogenous DNA and results in the reduced accumulation of RNA molecules with sequence similarity to the introduced nucleic acid (1, 2).
PTGS in plants can be suppressed by several virus-encoded proteins (6) and is closely related to RNA-mediated virus resistance and cross-protection in plants (7,8). Therefore, PTGS may represent a natural antiviral defence mechanism and transgenes might be targeted because they, or their RNA, are perceived as viruses. PTGS could also represents a defence system against transposable elements and may function in plant development (9-11). To account for the sequence specificity, and post-transcriptional nature of PTGS it has been proposed that antisense RNA forms a duplex with the target RNA thereby promoting its degradation or interfering with its translation (12).
One problem which exists in actually utilising efficient gene silencing, for instance via anti-sense mechanisms, is selecting appropriate regions to target. This problem has been reviewed in the literature (see Szoka (1997) Nature Biotechnology 15: 509; Eckstein (1998) Nature Biotechnology 16: 24). Proposed solutions to selecting good target regions include computational analysis (Patzel and Sczakiel(1998) Nature Biotechnology 16: 64-68) or Rnase H cleavage using chimeric anti-sense oligonucleotides (see Ho (1996) Nucleic Acid Res 24: 1901-1907; Ho et al (1998) Nature Biotechnology 16: 59-62). Other groups have used wide array of oligonucleotides to select those which form heteroduplexes (see Milner et al (1997) Nature Biotechnology 15: 537-541).
The present inventors have investigated PTGS of target genes initiated by a variety of silencing mechanisms in different organisms, and have established that in every case a previously uncharacterised species of antisense RNA complementary to the targeted mRNA was detected. These RNA molecules were of a uniform length, estimated at around 25 nucleotides, and their accumulation required either transgene sense transcription or RNA virus replication. Corresponding sense RNA molecules were also detected.
There have been no previous reports of such short sense and antisense RNA molecules (hereinafter, collectively, SRMs) that are detected exclusively in organisms exhibiting PTGS, possibly because (owing to their size) they could not have been readily detected by routine RNA analyses.
It appears that the SRMs may be synthesized from an RNA template and represent a specificity determinant and molecular marker of PTGS. Because of their correlation with PTGS and the nature of the molecules (short complementary molecules which could base pair with the target RNAs) they are believed to represent a signal and/or inducer or activator of PTGS.
The identification of this species by the present inventors may be utilised by those skilled in the art in a variety of methods and processes which are discussed in more detail below. Generally speaking the present invention provides, inter alia, methods of identifying and screening for gene silencing and particular silenced genes in organisms; processes for producing or isolating silencing agents, and such isolated agents themselves; methods for selecting target regions of nucleic acids which it is desired to silence and methods for silencing target genes using the agents or target regions generated as above. Also included are relevant materials. (e.g. nucleic acids, constructs, host cells, transgenic plants, silenced organisms) and methods of use of these.
Importantly, the disclosure herein provides evidence that SRMs may be a common mediator of PTGS in both plants and higher organisms, such as the nematode discussed in the Examples hereinafter. It was previously known that double stranded RNA induces a similar effect to plant PTGS in nematodes, insects (4) and protozoa (5). For instance PTGS has been demonstrated in Caenorhabditis elegans (a nematode worm) using DsRNA introduced into the worms by microinjection, imbibing or by allowing the worms to eat bacteria (E. coli) which are synthesizing dsRNA. There was also some evidence that in some examples of PTGS in plants and dsRNA interference in nematodes, a signal is produced which spreads and amplifies the silencing beyond the point of introduction of the original inducer of silencing. Although there were known to be certain similarities between the DsRNA induced silencing in nematodes and the causes of PTGS in plants, there was no clear evidence that the two are related.
Aspects of the invention will now be discussed in more detail.
Thus in one aspect of the present invention there is provided a method of detecting, diagnosing, or screening for gene silencing in an organism, which method comprises the steps of:
(i) obtaining sample material from the organism,
(ii) extracting nucleic acid material therefrom,
(iii) analysing the extracted nucleic acid in order to detect the presence or absence of SRMs therein,
The result of the analysis in step (iii) may be correlated with the presence of silencing in the organism.
The xe2x80x98samplexe2x80x99 may be all or part of the organism, but will include at least some cellular material.
The term xe2x80x98SRMsxe2x80x99 is used to describe the short RNA molecules described herein which are approximately 25 nucleotides in length. The size appears to be very characteristic, being estimated as approximately 25 nucleotides in all the cases tested (relative to the same molecular size markers when assessed by chromatography). However, it may be slightly more or less than this characteristic length (say plus or minus 1, 2, 3, 4 or 5 nucleotides) and where the term xe2x80x9825 nt RNAxe2x80x99 is used herein, it will be understood by those skilled in the art that the comments would apply equally in the event that the SRMs do not have this precise length.
Indeed the precise length may not be important, since the disclosure herein permits the identification, isolation and utilisation of SRMS in any case.
In performing the invention, it may be preferred to analyse or otherwise utilise short anti-sense RNA molecules (SARMs) rather than short sense RNA molecules (SSRMs). Nonetheless, where reference is made herein to SARMs (except where context clearly suggests otherwise) it will be appreciated by those skilled in the art that the SSRMs may also be used.
In particular, the SRMs methodology may be used as an indicator of PTGS. As is well known to those skilled in the art, PTGS occurs post-transcriptionally: i.e. the transcription rates of the suppressed genes are unaffected. The term xe2x80x98genexe2x80x99 is used broadly to describe any sequence which is suitable for translation to a protein.
Thus the presence of SRMs can be used as a diagnostic test for the existence of PTGS.
In one embodiment of this-aspect there is disclosed a method of detecting or identifying the silencing of a target gene in an organism, which method further comprises characterising any SRMs which are present. It should be noted that PTGS effects are very dominant. In principle the presence of SRMs may indicate the silencing of more than one gene, providing that they have sufficient homology.
xe2x80x98Characterisedxe2x80x99 and xe2x80x98characterisingxe2x80x99 does not necessarily imply complete sequencing, although this may be preferred. In order to detect silencing of a known sequence, the SRMs may be fully or partially sequenced, or sequence identity or similarity may be inferred from e.g. blotting.
Applications for such a diagnostic test will depend on the organism in question. For instance, in plants, since PTGS is the basis for a lot of pathogen derived resistance (PDR), GM field crops (e.g. individuals, or populations) engineered for PDR could be monitored xe2x80x9cin fieldxe2x80x9d by checking for the existence of 25 nt RNA to make sure that the PDR was still operating prior to the attack by the virus.
Similarly, crops depending upon co-suppression for the knockout of a particular plant gene to achieve a specific modified trait could be assayed for the continued function of PTGS by checking the presence of 25 nt RNA against the intended target. Such an assay may be particularly useful in view of evidence that transgenes have a tendency to become transcriptionally inactivated over the generations. PTGS depends upon transcription of the initiating transgene to function and so if this gets reduced the PTGS will begin to fail. Monitoring 25 nt RNA provides a quick way to test the lines. Non-limiting Examples of silenced genes which could be monitored in this way include any of those which have already been shown to be suppressible by PTGS in the literature. These may include, for example, chalcone synthase of petunia or polygalacturonase of tomato (Jorgensen, R. A. (1995), Science, 268: 686-691, Hamilton, A. J., et al (1995), Current Topics In Microbiology and Immunology, 197: 77-89).
It is also possible that the process of PTGS underlies certain plant developmental processes. If there are plant genes which are being targeted naturally as a result of PTGS in order to satisfy some plant developmental programme, a 25 nt RNA corresponding to sequences from these genes may be detectable.
Thus, in this embodiment, the SRMs may be used to identify and isolate an unknown target. This could be achieved by analysing the 25 nucleotide fraction of RNA from a plant, tagging it with a marker (e.g. a radioactive one) and then using this radioactive RNA to probe a library of plant genes. This probe, will anneal to genes which are undergoing PTGS in the plant, which genes can then be further analysed or characterised if required. Such genes, inasmuch as they are novel, represent a further aspect of the present invention.
In a further aspect of the present invention, there is disclosed a process for producing or isolating short RNA molecules. As discussed above, SRMs may not be readily detected by routine RNA analyses, particularly those which include a step in which such molecules are xe2x80x98lostxe2x80x99 (for instance SRMS may not be efficiently retained on silica columns which are used to isolate longer molecules such as mRNAs). A preferred process is set out in the Examples hereinafter. Broadly speaking, the processes provided divide into two parts: extraction/purification and detection.
For extraction, initial steps may be performed using conventional RNA extraction methods and kits appropriate to the organism in question, modified as required to ensure that SRMs are retained at each step.
In order to enhance purification of short RNAs, the extraction may optionally be followed by one or more of the following steps:
(i) filtration (e.g. through Centricon 100 concentrators (Amicon) or similar),
(ii) differential precipitation (e.g. with 5% polyethylene glycol(8000)/0.5M NaCl)
(iii) ion exchange chromatography (e.g. using Qiagen columns).
These steps enrich and purify the short RNAs to greater degrees than is obtained with the routine rRNA extraction method alone, and may be performed in conventional manner using, if required, proprietary reagents.
It should be noted that there is no requirement that the short RNAs be purified to homogeneity, provided only that they are capable of detection.
Regarding detection, because of their small size the method for this is not the usual one for xe2x80x9cRNA gel blot analysisxe2x80x9d although the principle is the same i.e. separation of the RNA molecules according to size by electrophoresis through a gel.
Preferably the gel is a 15% polyacrylamide gel containing 7M urea as a denaturant and TBE (0.5xc3x97) as a buffer.
The RNAs are preferably transferred to a hybridisation membrane by electrophoresis (rather than the more conventional capillary blot). Once the RNA is on the membrane, it is covalently attached to it by UV irradiation. The membrane is then placed in xe2x80x9cprehybridisation solutionxe2x80x9d for a short time.
Radioactive probe may be prepared using standard techniques. However, preferably, it is made as a single stranded RNA molecule transcribed in vitro from an appropriate plasmid DNA templates. The length of the probe may, preferably, be shortened by limited hydrolysis before adding to the prehybridisation solution; this may reduce non-sequence specific binding of probe to the membrane.
The hybridisation of the probe to its target is allowed to proceed at a stringency level (specific temperature, salt concentration and the concentration of formamide in the prehybridisation solution) appropriate to the requirements of the process. For instance low temperature, high salt, no formamide equals a low stringency, which may permit short probes or probes with imperfect homology to the target to hybridise with the target. Conversely high temperature, low salt and formamide mean high stringency with only lengthy duplexes stable under these conditions. Preferred conditions are 45% formamide, 7% SDS, 0.3M NaCl, 0.05M Na2HPO4/NaH2PO4 (pH7), 1xc3x97Denhardt""s solution, and single stranded heterologous nucleic acid (e.g. derived from salmon sperm).
This is one preferred process of purifying (or partially purifying) SRMs for the purpose of detection and/or further characterising e.g. for sequencing. However it should be understood that the present invention is in no way limited to this particular format, and others methods for SRMs analysis, such as those which may be devised in the future, will also be encompassed.
The process described above may form part of a more extensive process for producing or isolating a silencing agent for a target gene, which silencing agent is a preferably a SRM, the process comprising the steps of:
(i) silencing a target gene in an organism,
(ii) performing a process as described above in order to isolate a SRM appropriate for that gene.
xe2x80x98Silencing agentxe2x80x99 in this context may be one or more of an inducer, signal, or specificity determinant of gene silencing, particularly PTGS. Preferably this will be a SARM (as opposed to a SSRM). Isolated silencing agents obtained or obtainable by this method, inasmuch as they are novel, form a further aspect of the present invention.
The initial silencing step may be achieved by any conventional method appropriate to the organism in question. For instance in plants it could be by silencing of endogenous, homologous genes (co-suppressionxe2x80x94see, for example, van der Krol et al., (1990) The Plant Cell 2, 291-299; Napoli et al., (1990) The Plant Cell 2, 279-289; Zhang et al., (1992) The Plant Cell 4, 1575-1588, and U.S. Pat. No. 5,231,020). Further refinements of the gene silencing or co-suppression technology may be found in WO95/34668 (Biosource); Angell and Baulcombe (1997) The EMBO Journal 16,12:3675-3684; and Voinnet and Baulcombe (1997) Nature 389: pg 553 (systemically induced transgene silencing). Other options include transgene silencing; RNA mediated defence against viral infection, and transgenic, homology-dependent, virus resistance, or use of dsRNA in the case of nematodes.
In a further aspect of the present invention there is disclosed a method for identifying or selecting a target region of a gene, which gene it is desired to silence, which method comprises:
(i) silencing the target gene in an organism,
(ii) performing a process as described above in order to isolate a SRM appropriate for that gene,
(iii) identifying a region in the sequence of the gene which corresponds to the sequence of the SRM.
The region may identified most readily by comparing the sequence of the SRM with the sequence of the gene; however any appropriate method may be used (e.g. RNAase protection). If several SRMs are isolated, then several target regions may be identified.
As described in the introduction, this method provides an alternative to e.g. computational analysis in order to identify the most suitable site on e.g. an mRNA corresponding to a target gene, for targeting for silencing e.g. with an antisense construct. With the information obtained using the methods and processes herein about, more efficient antisense reagents (not necessarily RNAs) may be produced which are tailored such that they would be recognised and used by the PTGS machinery of the organism.
In a further aspect of the present invention there is disclosed a method of silencing a target gene in an organism which utilises the methodology described above.
xe2x80x9cSilencingxe2x80x9d in this context is a term generally used to refer to suppression of expression of a gene. The degree of reduction may be so as to totally abolish production of the encoded gene product, but more usually the abolition of expression is partial, with some degree of expression remaining. The term should not therefore be taken to require complete xe2x80x9csilencingxe2x80x9d of expression. It is used herein where convenient because those skilled in the art well understand this.
In one embodiment, the method comprises introducing anti-sense molecules [SARMs] appropriate for the target gene into the organism in order to induce silencing. This could be done, for instance, by use of transcribable constructs encoding the SARMs.
In a related embodiment, the silencing may be achieved using constructs targeting those regions identified by the SRMs-based method disclosed above. Such constructs may e.g. encode antisense oligonucleotides which target all are part of the identified region, or a region within 1,2,3,4,5,10, 15 or 20 nucleotides of the identified region.
Suitable target genes for silencing will occur to those skilled in the art as appropriate to the problem in hand. For instance, in plants, it may be desirable to silence genes conferring unwanted traits in the plant by transformation with transgene constructs containing elements of these genes. Examples of this type of application include silencing of ripening specific genes in tomato to improve processing and handling characteristics of the harvested fruit; silencing of genes involved in pollen formation so that breeders can reproducibly generate male sterile plants for the production of hybrids; silencing of genes involved in lignin biosynthesis to facilitate paper making from vegetative tissue of the plant; silencing of genes involved in flower pigment production to produce novel flower colours; silencing of genes involved in regulatory pathways controlling development or environmental responses to produce plants with novel growth habit or (for example) disease resistance; elimination of toxic secondary metabolites by silencing of genes required for toxin production. In addition, silencing can be useful as a means of developing virus resistant plants when the transgene is similar to a viral genome.
As described above, the disclosure herein provides evidence that SRMs may be a common mediator of PTGS in both plants and higher organisms. These new findings can be utilised, inter alia, in that it now appears that induction of SRMs (particularly SARMs) with an appropriate specificity in one organism (say, a plant) may be used to silence an appropriate target gene in another organism (say, a predator) which comes into contact with that plant.
In one aspect of the invention there is provided a method for targeting a gene in a first organism, which method comprises generating a SARMs silencing agent in a second organism, and introducing the SARMs into the first organism.
The SARMs may be generated by any appropriate silencing method. Preferably the target gene will be one which is not an endogenous gene in the second organism (but preferably is endogenous to the first). The xe2x80x98contactxe2x80x99 may be ingestion, injection, or any other method of administration. How, precisely, the method is performed will depend on the organisms and genes involved.
For instance, in the case of plants and plant predators, it is known that the systemic signal of PTGS travels out of plant cells into the phloem (sap) of plants and induces silencing in previously non-silencing parts of the plant. In the light of the present disclosure it is clear that, since plant parasitic nematodes feed directly upon the sap and contents of plant cells, they will ingest the signal and inducer of PTGS (i.e. SARMs) in the plant.
As shown in the Examples below, it appears that the same type of SARMs are present in C. elegans which are undergoing PTGS induced by the ingestion of dsRNA. This implies that the mechanism of PTGS in plants and nematode is similar if not identical. Thus plant SARMs may trigger the PTGS of any similar sequences present in the worm. Therefore when the nematode feeds on the plant, and eats the PTGS signal, if there is homology between the plant""s transgene from which the PTGS signal derived and a nematode gene, PTGS of that gene ought to be triggered in the worm.
Where the targeted gene is an essential gene, this method provides a means of controlling or killing plant predators or pests. Naturally, more than one gene can be targeted at once.
It may be desirable that the targeted gene is one which is either not present, or not important, in the wild-type plant or other potential consumers of the plant i.e. is nematode specific gene, such as a nematode protease gene. This gives the method a high degree of specificity.
Interestingly C. elegans is a nematode distantly related to the nematodes that parasitise plants. Since dsRNA induced-PTGS is conserved between nematodes, protozoa and insects it is likely that these other organisms which support PTGS may be susceptible to SARMs.
DsRNA interference has also been shown to work in insects and transgene induced PTGS works in fungi, so it is likely that this is a mechanism that is broadly conserved across the kingdoms. This implies that any organism that directly feeds off plant cellular contents or extracellular components such as sap could ingest PTGS specific SARMs. If these have sequence homology with genes resident in the parasite, PTGS of these genes could be initiated.
Thus insect specific genes (e.g. from aphids) represent a further target. Most preferable would be those insect genes or sequences not found in beneficial insects, such a ladybirds.
Other targets include genes specific for plant parasites of plants which feed off the host plant.
Specifically regarding higher animals (e.g. mammals, fish, birds, reptiles etc.) methods of the present invention include, inter alia:
(i) methods for detecting or diagnosing gene silencing, or silencing of particular genes, in the animal by using SRMs as described above,
(ii) methods for identifying silenced genes in the animal by using SRMs as described above,
(iii) methods for selecting target sites on genes to be silenced using SRMs as described above,
(iv) method for silencing a target gene in the animal, either directly, or through an animal-derived transgene in a second organism (e.g. a plant) as described above.
Generally speaking target genes in animals may be those whose functional impairment beings therapeutic benefits. Typical genes of interest may be (for instance) those involved w apoptosis, cancer, cell-cycle regulation, neurological processes, signal transduction etc. Examples and references can be found in the Oncogene Research Products 1999 General Catalog, pp 21-265, available from Oncogene Research Products, 84 Rogers Street, Cambridge, Mass. 02142, U.S. Preferred examples include oncogenes, transcriptional regulators, pocket proteins, members of the MHC superfamily (to produce allotypic organs) etc.
Some further aspects and applications for the present invention will now be discussed.
According to one aspect of the present invention there is provided, preferably within a vector suitable for stable transformation of a plant cell, a DNA construct in which a promoter is operably linked to DNA for transcription in a plant cell to generate either:
(i) a SARM as described above, or
(ii) an anti-sense RNA molecule selected to target a region identified by the SRM-based methods discussed above.
Generally speaking, such constructs may be used to silence genes within plants, or within organisms predating or being administered material from plants, in the terms discussed above.
Anti-sense partial gene sequences selected in accordance with SRM-based methods may be used analogously to those previously used in the art. See, for example, Rothstein et al, 1987; Smith et al, (1988) Nature 334, 724-726; Zhang et al, (1992) The Plant Cell 4, 1575-1588, English et al., (1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Bourque, (1995), Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496. Generally the selected sequence will be less than 50, 40, 30, 25, or 20 nucleotides. It may be preferable that there is complete sequence identity in the targeting (e.g. foreign) sequence in the construct and the target sequence in the plant, although total complementarity or similarity of sequence is not essential.
Again, generally speaking, plants and associated methods and processes which form a part of the present invention are either those which:
(i) are transformed with the xe2x80x98targetingxe2x80x99 anti-sense vectors such as those described above, for instance so as to silence an (endogenous) target gene in the plant or perhaps a viral gene,
(ii) are transformed with transgenes taken from other organisms such as to induce transgene silencing and thereby generate SARMs which can be used to silence a target gene in that other organism, or
(iii) are transformed with vectors which encode SARMs directly, which can be used for either purpose.
The general methodology discussed below will be applicable to all of these applications.
A vector which contains the construct may be used in transformation of one or more plant cells to introduce the construct stably into the genome, so that it is stably inherited from one generation to the next. This is preferably followed by regeneration of a plant from such cells to produce a transgenic plant. Thus, in further aspects, the present invention also provides the use of the construct or vector in production of a transgenic plant, methods of transformation of cells and plants, plant and microbial (particularly Agrobacterium) cells, and various plant products.
The function of the promoter in the construct is to ensure that the DNA is transcribed into RNA containing the viral sequences. By xe2x80x9cpromoterxe2x80x9d is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3xe2x80x2 direction on the sense strand of double-stranded DNA). A promoter xe2x80x9cdrivesxe2x80x9d transcription of an operably linked sequence.
xe2x80x9cOperably linkedxe2x80x9d means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
Preferred promoters may include the 35S promoter of cauliflower mosaic virus or the nopaline synthase promoter of Agrobacterium tumefaciens (Sanders, P. R., et al (1987), Nucleic Acids Res., 15: 1543-1558). These promoters are expressed in many, if not all, cell types of many plants. Depending on the target gene of amplicon gs, other promoters including those that are developmentally regulated or inducible may be used. For example, if it is necessary to silence the target gene specifically in a particular cell type the construct may be assembled with a promoter that drives transcription only in that cell type. Similarly, if the target gene is to be silenced following a defined external stimulus the construct may incorporate a promoter that is be activated specifically by that stimulus. Promoters that are both tissue specific and inducible by specific stimuli may be used. Suitable promoters may include the maize glutathione-S-transferase isoform II (GST-II-27) gene promoter which is activated in response to application of exogenous safener (WO93/01294, ICI Ltd).
An additional optional feature of a construct used in accordance with the present invention is a transcriptional terminator. The transcriptional terminator from nopaline synthase gene of agrobacterium tumefaciens (Depicker, A., et al (1982), J. Mol. Appl. Genet., 1: 561-573) may be used. Other suitable transcriptional terminators will be well known ot those skilled in the art.
Those skilled in the art are well able to construct vectors (including those based on xe2x80x98nakedxe2x80x99 DNA) and design protocols for recombinant gene expression. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley and Sons, 1992.
Specific procedures and vectors previously used with wide success upon plants are described by Bevan, Nucl. Acids Res. (1984) 12, 8711-8721), and Guerineau and Mullineaux, (1993). Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed). Oxford, BIOS Scientific Publishers, pp 121-148.
For introduction into a plant cell, the nucleic acid construct may be in the form of a recombinant vector, for example an Agrobacterium binary vector. Microbial, particularly bacterial and especially Agrobacterium, host cells containing a construct according to the invention or a vector which includes such a construct, particularly a binary vector suitable for stable transformation of a plant cell, are also provided by the present invention.
Nucleic acid molecules, constructs and vectors according to the present invention may be provided isolated and/or purified (i.e. from their natural environment), in substantially pure or homogeneous form, or free or substantially free of other nucleic acid. Nucleic acid according to the present invention may be wholly or partially synthetic. The term xe2x80x9cisolatexe2x80x9d encompasses all these possibilities.
An aspect of the present invention is the use of a construct or vector according to the invention in the production of a transgenic plant.
A further aspect provides a method including introducing the construct or vector into a plant cell such that the construct is stably incorporated into the genome of the cell.
Any appropriate method of plant transformation may be used to generate plant cells containing a construct within the genome in accordance with the present invention. Following transformation, plants may be regenerated from transformed plant cells and tissue.
Successfully transformed cells and/or plants, i.e. with the construct incorporated into their genome, may be selected following introduction of the nucleic acid into plant cells, optionally followed by regeneration into a plant, e.g. using one or more marker genes such as antibiotic resistance. Selectable genetic markers may be used consisting of chimeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate.
When introducing a nucleic acid into a cell, certain considerations must be taken into account, well known to those skilled in the art. The nucleic acid to be inserted should be assembled within a construct which contains effective regulatory elements which will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material should occur. Finally, as far as plants are concerned the target cell type must be such that cells can be regenerated into whole plants.
Plants transformed with the DNA segment containing the sequence may be produced by standard techniques which are already known for the genetic manipulation of plants. DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-01167181 NAR 12(22) 8711-87215 1984), particle or microprojectile bombardment (U.S. Pat. No. 5,100,792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al. (1987) Plant Tissue and Cell Culture, Academic Press), electroporation (EP 290395, WO 8706614 Gelvin Debeyserxe2x80x94see attached) other forms of direct DNA uptake (DE 4005152, WO 9012096, U.S. Pat. No. 4,684,611), liposome mediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol. 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U.S.A. 87: 1228 (1990d). Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech. Adv. 9: 1-11.
Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (Toriyama, et al. (1988) Bio/Technology 6, 1072-1074; Zhang, et al. (1988) Plant Cell Rep. 7, 379-384; Zhang, et al. (1988) Theor Appl Genet 76, 835-840; Shimamoto, et al. (1989) Nature 338, 274-276; Datta, et al. (1990) Bio/Technology 8, 736-740; Christou, et al. (1991) Bio/Technology 9, 957-962; Peng, et al. (1991) International Rice Research Institute, Manila, Philippines 563-574; Cao, et al. (1992) Plant Cell Rep. 11, 585-591; Li, et al. (1993) Plant Cell Rep. 12, 250-255; Rathore, et al. (1993) Plant Molecular Biology 21, 871-884; Fromm, et al. (1990) Bio/Technology 8, 833-839; Gordon-Kamm, et al. (1990) Plant Cell 2, 603-618; D""Halluin, et al. (1992) Plant Cell 4, 1495-1505; Walters, et al. (1992) Plant Molecular Biology 18, 189-200; Koziel, et al. (1993) Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology 25, 925-937; Weeks, et al. (1993) Plant Physiology 102, 1077-1084; Somers, et al. (1992) Bio/Technology 10, 1589-1594; WO92/14828). In particular, Agrobacterium mediated transformation is now emerging also as an highly efficient transformation method in monocots (Hiei et al. (1994) The Plant Journal 6, 271-282).
The generation of fertile transgenic plants has been achieved in the cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994) Current Opinion in Biotechnology 5, 158-162.; Vasil, et al. (1992) Bio/Technology 10, 667-674; Vain et al., 1995, Biotechnology Advances 13 (4): 653-671; Vasil, 1996, Nature Biotechnology 14 page 702).
Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g. bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).
Following transformation, a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al., Cell Culture and Somatic Cel Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.
The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practicing the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.
Also according to the invention there is provided a plant cell having incorporated into its genome a DNA construct as disclosed. A further aspect of the present invention provides a method of making such a plant cell involving introduction of a vector including the construct into a plant cell. Such introduction should be followed by recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome. RNA encoded by the introduced nucleic acid construct may then be transcribed in the cell and descendants thereof, including cells in plants regenerated from transformed material. A gene stably incorporated into the genome of a plant is passed from generation to generation to descendants of the plant, so such descendants should show the desired phenotype.
The present invention also provides a plant comprising a plant cell as disclosed.
A plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders"" Rights.
In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed.
The present invention may be used in plants such as crop plants, including cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorgum, millet, cassava, barley, pea and other root, tuber or seed crops. Important seed crops are oil seed rape, sugar beet, maize, sunflower, soybean and sorghum. Horticultural plants to which the present invention may be applied may include lettuce, endive and vegetable brassicas including cabbage, broccoli and cauliflower, and carnations and geraniums. The present invention may be applied to tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum, poplar, eucalyptus and pine.
In relation to use in mammals or other higher animals, DNA vectors (including naked DNA suitable for expression in mammals) of the present invention encode either:
(i) a SARM as described above, or
(ii) an anti-sense RNA molecule selected to target a region identified by the SRM-based methods discussed above.
Such vector may be based on any appropriate vector known to those skilled in the art. For instance incorporation of this DNA into mammalian cells to produce such antisense RNA in vivo might be accomplished using vectors based on the disclosure of European patent application 909052736.3 (VICAL), HSV, vaccinia or adenovirus (see Principles of Gene Manipulation (1994) 5th Edit. Old and Primrose 5th Edition, Blackwell Scientific Publications). Viral vectors for use in gene therapy are discussed by Vile (1997) Nature Biotechnology 15; 840-841. A non-viral gene therapy approach is discussed by Sebestyen et al (1998) Nature Biotechnology 16: 80-85. The use of a variety of gene therapy delivery systems (including HSV VP22) is discussed by Fernandez and Baylay (1998) Nature Biotechnology 16: 418-420 and references therein.
Also provided by the present invention is an organism, preferably a non-human mammal, comprising cells in which a target gene is subject to PTGS by use of the SARM-based methods or materials disclosed herein. Particularly preferred is a rodent e.g. murine organism. In this embodiment the invention provides an alternative to known methods of producing xe2x80x98knock outxe2x80x99 mammals in which specific gene activities have been impaired (see e.g. Boerrigter et al (1995) Nature 377: 657-659, or Gossen and Vijk (1993) Trends Genet 9: 27-31.)
The invention will now be further described with reference to the following non-limiting Examples describing work of the inventors. The results are also discussed, and suggestions made as to the origin of the SRMs of the present invention. However it will be appreciated by those skilled in the art that the materials, methods and processes in the present disclosure may be usefully applied irrespective of the precise underlying mechanisms involved.
All references discussed herein, inasmuch as they may be required to supplement the present disclosure, are incorporated herein by reference.


A method for repairing concrete structural elements reinforced with steel rebar includes steps of: removal of debris and rust; attachment of expanded mesh zinc metal for sacrificial passive corrosion protection; and overwrapping with flexible panels of fiber-reinforced polymer composite material.

Most large concrete structures include a skeleton of welded steel rods for reinforcement. Because concrete is permeable by water, the steel rods eventually rust and corrode. The problem of corrosion of steel reinforcement is extreme in the case of a concrete column or similar structure that is partially submerged in seawater, such as a bridge piling; the salt ions aid corrosion and partial immersion in water helps drive electrochemical reactions, which are generally deleterious. Another significant source of corrosion of steel reinforcement is de-icing salt, which especially affects the deck of a bridge.
Corrosion of the steel is harmful to the structure. As the steel rods are dissolved or replaced by rust, they lose strength. Rust stains on the structure are ugly and may cause worry in persons using the structure. Corroded Steel has a greater volume than uncorroded steel; this expansion can crack the concrete and cause chunks to spall. Corrosion of the steel reinforcement can lead to eventual failure of the structure.
A widely used method of repairing cracked and spalled concrete structures, including bridge pilings, is to wrap structural elements in high-strength fiber-reinforced polymer composite panels. The wrap strengthens the structural element and partially shields it from further infiltration by water. A small amount of expansion of the steel due to residual corrosion slightly strengthens the composite wrap by putting it in tension. This method is discussed in more detail in U.S. Pat. No. 5,607,527, incorporated herein by reference.
In the case of structures in very corrosive environments, such as partly submerged in seawater, the composite wrap method does not protect the structure for as many years as is usually desired. Therefore, there is a need for a repair and protection method that has the many advantages of the composite wrap method, but that provides a longer reliable lifetime for structures in very corrosive environments.

Method for repairing steel-reinforced concrete structure


An electronic module has a heat sink with an upper surface and a lower surface, a plurality of leads arranged adjacent the heat sink and at least one circuit element with two vertical semiconductor power switches. The two vertical semiconductor power switches of each circuit element are arranged in a stack and are configured to provide a half-bridge circuit having a node defining an output. The first vertical semiconductor power switch of each of the circuit elements is mounted on the upper surface of the heat sink by an electrically conductive layer such that the lower surface of the heat sink provides the ground contact area of the electronic module.

Due into the increasing complexity of the current supply required by microprocessors in, for example, motherboards for computers and notebooks, multi-phase dc-dc buck converters are provided. A current of 2 to 20 amps can typically be provided by each phase of the buck converter. A buck converter typically comprises one or more half-bridge circuits, each provided by two power semiconductor power switches, such as power MOSFET devices, and further passive components, such as an inductance and a capacitance. The switching of the power MOSFET devices is controlled by a control semiconductor chip.
The components of the buck converter may be provided as individual components which are mounted on a printed circuit board. Alternatively, a number of components accommodated in a single housing to provide a multi-chip module, also referred to as a System-in-Package (SiP), which has the advantage that the assembly of the module on the board is simplified and the space required on the board can be reduced.
Due to the increasing demands for smaller components and higher power, insufficient heat dissipation from multi-chip modules may deleteriously affect the functionality of the module and/or neighboring components on the board. Improved heat dissipation from multi-chip modules is, therefore, becoming increasingly important.

Electronic module with stacked semiconductors


An automatic stacked parking or storage system includes a plurality of stationary docking cells, a plurality of carriages, and a central management system communicating with and controlling operations of the docking cells. Each docking cell has a ground driving device affixed on the bottom of the docking cell to drive a carriage horizontally in different directions of the docking cell, a docking cell control and interface unit (CIU), and one or more carriage position and ID sensor. A loaded carriage is moved horizontally by the ground driving devices of corresponding docking cells from a starting to a destination docking cell according to a route selected by a route optimization module. The location of the loaded carriage in the selected route is dynamically updated according to real time reports from each CIU of the corresponding docking cells. The system transports multiple loaded carriages at the same time for parking/storage and retrieval.

Space and land in urban are such a treasure, saving in space and construction cost is invaluable. In parking garages, recently most solutions are directed to providing stackable parking system in multi-level garages with elevators to replace conventional garages that have ramps and require larger and complex building structure.
Some stacked parking systems use horizontally moving-on-track lifts, or circular parking silo having a rotating vertical lift to transport cars into stacked bays. These systems have a high demand of lift usage, which relies on a lifting device for each parking and retrieval of a car. In these systems, at any time if the lifting device is out of service because of malfunction or maintenance, no parking or retrieving service can be provided by the system. Moreover, these existing systems have limited bays-per-lift and have space constrains to meet demands of a heavy traffic facility. Furthermore, these systems require fixed blueprints for the facility and have a low adaptability to available existing buildings or storage facilities.
Another type of system uses robotic or non-robotic shuttles to move cars to designated locations. This type of approach can provide dense parking. However, such a system is limited by the number of shuttles that can be used at the same time in order to prevent collision. It also has less systematic controlled movement, and less reliable battery powers in the shuttles. Therefore, shuttle operated systems have limited utility for high traffic capacity facilities.
Other known storage systems move carriers horizontally to designated locations within a facility configured as a matrix. One type system uses carriers having onboard side driving mechanism to drive one carrier against the next carrier to enable the movement. This type of system requires almost all storage spaces being occupied by carriers in order to support the carrier movement, due to the reliance on mutual engagement of neighboring units. Although such a system can support high density storage, such as for storage of payload, it is not suitable for heavy traffic facilities, because the extent of shuffling of the carriers is extensive in a process of moving one carrier from one location to another. Moreover, battery operated onboard driving mechanism requires frequent maintenance. Another type system uses combined upper and lower driving devices to move a carrier engaged between two sets of driving devices. Such system involves complex driving mechanisms and it relies on the driver of the overall system.
Therefore, there is a need for an improved stacked storage system that overcomes the above mentioned deficiencies. It is desirable for a system that is more versatile in its structure blueprint, and has improved operation efficiency and flexibility, and high tolerance to operating conditions of structural components of the system. It is desirable to have a parking system that can handle multiple parking and retrievals at the same time, which are particularly needed for high capacity and heavy traffic facilities.

Automatic stacked storage system for parking or storage


A disc recording and/or reproducing device includes a disc compartment that has a plurality of disc mounts placed one upon another, on which discs are mounted. The device also includes a recording and/or reproducing mechanism for selectively recording and/or reproducing a disc mounted on one of the plurality of disc mounts. An injection mechanism is included for ejecting the disc mounted on the disc mount. A control mechanism is included that moves along a line substantially perpendicular to the placing direction and is configured to start the recording and/or reproducing mechanism when in a first position and start the ejection mechanism when reaching a second position on another side of the line.

The present invention relates to a disc recording and/or reproducing device for receiving and recording/reproducing a plurality of discs.
There is a disc player by way of disc recording and/or reproducing device mounted on motor vehicles for recording and/or reproducing sound. The disc player is constructed to store a plurality of CDs (compact discs) in a magazine and change the whole CDs at a time together with the magazine. This leads to a device enlargement, so that the main body is received in a trunk, while only the part necessary for operation is arranged in a cabin. However, changing CDs is not possible during driving of the vehicle, and requires stop thereof to open the trunk, forming a bothersome affair.
On this account, vehicle-mounted disc players to be received in cabins are developed in recent years. Such vehicle-mounted disc player, which is required to enable storage of a plurality of CDs, is received in a predetermined in-dash space in the cabin. Accordingly, there remains an essential challenge of a reduction in size of the vehicle-mounted disc players.
The vehicle-mounted disc player includes a roughly rectangular-prism casing of a given dimension called 1 DIN size or the like, in which, for example, six trays for individually mounting six CDs thereon are placed one upon another to constitute a disc compartment. Six CDs can individually be loaded and ejected to successively select one of six CDs for reproduction.
A prior-art reference 1 of the vehicle-mounted disc player is constructed such that for selecting and reproducing one of six CDs, six trays for mounting CDs are arranged to be placed vertically one upon another, and a male screw having a greater pitch in the middle portion along the length is formed with a shaft arranged through the trays to obtain a screwed shaft. Rotation of the screwed shaft causes the trays to successively rise from the top. When stopping rotation of the screwed shaft with a selected tray mounting selected CD being located in the middle portion of the screwed shaft, spaces are defined above and below the selected tray. Then, a spindle motor and turntable and a pickup unit are inserted below selected CD to engage a claw protruding upwardly from the center of the turntable with a hole of CD through upward motion of the turntable, mounting CD on the turntable. After withdrawing the turntable, CD is rotated for reproduction.
A prior-art reference 2 is constructed such that after moving CD to be reproduced to a predetermined level position by moving a cartridge upwardly or downwardly, spaces are formed above and below CD to be reproduced by space forming means. A turntable and a chucking plate are introduced into the spaces to chuck CD to be reproduced on the turntable, and an optical pickup is moved from a standby position to a reproduction position.
However, the prior-art reference 1 presented the following problem:
Since the device is not configured to hold CD from both sides for rotation, coupling is not sufficient between the turntable and CD to deteriorate the vibration characteristics during reproduction. In some cases, CD can accidentally be detached from the turntable.
The prior-art reference 2 presented the following problems 1) and 2):
1) The cartridge is moved upwardly or downwardly with respect to the optical pickup fixed at the level position shown by H in FIG. 38, so that for moving the cartridge 201 as illustrated by the solid line from the state of reproducing CD 202 on the uppermost stage to the state of reproducing CD 203 on the lowermost stage of the cartridge 201, the cartridge 201 should be raised to the position as illustrated by the two-dot chain line. As a result, a height T1 of a cartridge receiving space is roughly twice as great as a height T2 of the cartridge 201, forming a bottleneck to a reduction in thickness of the disc player.
2) After movement of CD to be reproduced to a predetermined level position, the space forming means are moved toward the CD to form spaces above and below CD to be reproduced. Turntable and chucking plate are introduced into the spaces to chuck CD on the turntable, and the optical pickup is moved from the standby position to the reproduction startup position. Thus, changing CD requires a lot of time.
Additionally, for accommodation of a plurality of CDs, the device needs to hold parts of operation mechanisms for carrying out various operations such as CD loading, ejection, recording, reproduction, etc., leading to difficult reduction in size of the device in its entirety.
Therefore, the present invention aims to provide a disc recording and/or reproducing device that solves such problems.
A disc recording and/or reproducing device is constructed such that it comprises: a disc compartment comprising a plurality of disc mounting means placed one upon another, on which discs are mounted; a recording and/or reproducing means for selectively recording and/or reproducing a disc mounted on a selected one of said plurality of disc mounting means; an ejection means for ejecting the disc mounted on said disc mounting means; a control means, which moves along a line substantially perpendicular to said placing direction, for starting said recording and/or reproducing means when reaching a first position on one side along the line, and said ejection means when reaching a second position on another side along the line.
The disc recording and/or reproducing device further comprises a space forming means for moving in the placing direction the disc mounting means in said disc compartment and adjacent to the selected disc mounting means so as to form a first space on one side of the placing direction of the selected disc mounting means, wherein said recording and/or reproducing means ensures action of moving a disc driving means for holding and rotating/driving the disc to the first space formed by said space forming means, and wherein said ejection means ensures action of opening a shutter of a disc loading/unloading opening and moving the disc to said loading/unloading opening.
The disc recording and/or reproducing device is constructed such that said control means comprises a startup plate having a first engagement engaging with said recording and/or reproducing means and a second engagement engaging with said ejection means.
The disc recording and/or reproducing device is constructed such that said control means comprises a startup plate having a first engagement engaging with said recording and/or reproducing means and a second engagement engaging with said ejection means.
According to the invention, contradictory action of the recording and/or reproducing means and the ejection means can be ensured by the only one control means (startup plate), so that there is no need to separately arrange a startup means for starting the recording and/or reproducing means and a startup means for starting the ejection means, obtaining simplified device configuration and reduced number of parts, resulting in a reduction in size of the entire device.

Disk recording and/or reproducing device with starting and ejection control means


A ferrule includes a ferrule matrix, an optical fiber, and a dielectric reflective film. The optical fiber is disposed in an accommodating through hole of the ferrule matrix, and the dielectric reflective film covers an optical transmission surface of the optical fiber and a surface that is of the ferrule matrix and that faces a matching ferrule. The dielectric reflective film has a through hole, such that the dielectric reflective film does not shield a main optical path area of the optical transmission surface of the optical fiber. A reflective band of the dielectric reflective film includes at least a part of a communication band of the optical fiber. In this way, when light from the matching ferrule is transmitted to the ferrule matrix and the optical fiber, the dielectric reflective film reflects the light.

In a big data era, optical communication is widely used for transmitting massive data. However, an energy density of an optical transmission medium (for example, a single-mode fiber or a waveguide) is very high due to a rapid increase of data transmission amount. The optical transmission medium is a part of a ferrule of a connector and is disposed in a ferrule matrix. By connecting ferrules of two connectors, optical communication is achieved using optical transmission mediums in the two ferrules. Light from an optical transmission medium of one ferrule is transmitted to a surface of a ferrule matrix of the other ferrule. The light is absorbed by the surface and converted to heat. When heat accumulates, the ferrule matrix of the other ferrule is burnt down.

Ferrule, optical connector, optical communication element, communications device, and preparation method


The present invention relates to a dimensionally stable oxygen-evolving anode for use in an electrolytic cell for the production of aluminium. The anode comprises of a container made from an alloy comprising aluminium and at least one metal more noble than aluminium; a fluid bath in the bottom of the container having the ability to dissolve aluminium, said fluid having a density that is higher than the density of molten aluminium at the operating temperature of the cell, a pool of molten aluminium floating on top of the fluid bath in the bottom of the container; a refractory layer arranged on the inner sidewalls of the container at least in the area of the pool of molten aluminium, said refractory layer protecting the molten aluminium from contacting the inner sidewalls of the container.

Electrolytic production of aluminium using the Hall-Héroult electrolytic process is well known. In the Hall-Héroult process aluminium is produced from Al2O3 dissolved in an electrolytic bath of molten cryolite and AlF3 at a temperature of about 960° C. using carbon anodes. Aluminium ions are reduced to aluminium at the cathode while oxygen is combined with carbon at the anode to form CO2 gas.
In this process about half a kilogram of carbon is consumed for each kilogram of produced aluminium. Carbon anodes must therefore routinely be replaced. The CO2 gas produced at the anode is considered to be a green house gas, and is an undesired by product of the process.
Efforts have been made to provide inert and dimensional stable anodes for use in the electrolytic production of aluminium. Most of the research has concentrated in oxide-based ceramic anodes and cermet anodes. However, these efforts have so far not been commercialised for production of aluminium.
U.S. Pat. No. 5,254,232 describes an anode for use in molten electrolyses of metals such as aluminium. The anode comprises an alloy of the product metal such as aluminium and a more noble metal upon which is formed an oxide of the product metal as a protective layer. For electrolysis of aluminium the protective layer consists of Al2O3. All surfaces of the anode intended to be in contact with the electrolyte in the cell have this protective layer. The anode of U.S. Pat. No. 5,254,232 suffers from the disadvantages that the protective layer may be dissolved, particularly if the content of alumina in the electrolyte becomes low. Thus one has to operate the electrolytic cell with an electrolyte which is saturated with alumina. This can cause operational problems such as accumulation of undissolved alumina at the bottom of the electrolytic cell and will further provide problems in controlling the cell operation. If the protective layer of the anode is dissolved, the alloy of which the anode is made can be consumed resulting in failure of the anode.
U.S. Pat. No. 6,083,362 describes an anode for use in electrolytic production of aluminium, where the anode comprises a substrate made from a metal alloy defining a cavity where the substrate comprises an interior wall and an exterior surface where the substrate is capable of diffusing aluminium from the cavity to the exterior surface to provide a film covering portions of the exterior surface and mean for replenishing the film. The means for replenishing the film on the exterior surface of the substrate is a molten salt containing aluminium and an anion selected from the group consisting of a fluoride, a carbonate, a chloride, an oxide and combination of these.
The anode described in U.S. Pat. No. 6,083,362 has some disadvantages. The aluminium in the fluid salt composition is depleted as the aluminium diffuses through the substrate to the exterior surface. Thus, the concentration of aluminium in the salt varies and additional aluminium must be supplied to the molten salt in the cavity from time to time during the use of the anode to maintain the concentration of aluminium in the fluid molten salt bath in the cavity. Such periodic additions of aluminium to the salt is impractical in a commercial operation where the electrolytic cell is closed. Furthermore, variation in the aluminium content of the salt will cause variation in the composition and thickness of the protective layer and have a deleterious effect on the operation of the electrolytic cell. Finally as the aluminium content in the salt bath decreases it will be difficult to maintain a homogeneous concentration of aluminium in the salt bath. This may cause a too low aluminium activity locally in the salt bath resulting in a too low diffusion rate of aluminium through the metal alloy which may cause permanent changes locally in the metal alloy making it impossible to maintain the protective layer locally on the outside of metal alloy. It should be appreciated that no stirring device or other means for maintaining a homogeneous aluminium concentration in the salt bath is described in U.S. Pat. No. 6,083,362.
There is a need for commercial process for the production of aluminium using a dimensionally stable anode where the protective layer is stable.


A mixer includes an agitating means and a material scraping means in a spherical casing. A mixer further includes a cracking-dispersing means for an easily cohesive powder or an impalpable powder. If crushing of particles need be restricted to a moderate level, the mixer includes either one the above mixers as a mixer main body and an angle setting means for setting the tilt the mixer main body at a given angle.. The above mixers may preferably be used for agitating and mixing a powdery or granular material of all kinds, particle sizes, and specific gravities ranging from an easily cohesive powder or an impalpable powder to particles with which it is preferable to restrict a crushing level.

1. Field of the Invention
This invention relates to a batch-type mixer for agitating and/or mixing two or more powdered to particulate materials with one another or one or more powdered to particulate materials and a liquid with one another at an elevated speed and occasionally with moderate suppression of the crushing action.
2. Disclosure of the Prior Art
The mixing systems for powdered to particulate materials include a mixed system employing a mixer with a revolving vessel, and a batch- or continuous type mixer with a fixed vessel. For agitating and/or mixing powdered to particulate materials at an elevated speed, a high-speed batch type mixer with a fixed vessel is preferentially employed.
With the high-speed mixer, one or more kinds of powdered to particulate materials to be mixed together are injected in predetermined amounts into a fixed vessel and the agitating blades of various profiles mounted in the vessel are revolved at an elevated speed of the order of 500 to 10,000 rpm to fluidize and agitate the materials compulsorily to effect the mixing. This high speed mixer may be typified by a Henschel mixer, a high-speed mixer or a fine mixer.
It is required of the high speed mixer to elevate the mixing degree. The powdered to particulate materials may be classified into those having finer particle size, referred to hereafter as the powders or fine powders, or powdered or finely powdered materials, and those having coarser particle size, referred to hereafter as the particulate materials. The powdered and finely powdered materials tend to be flocculated and, when they are mixed in the flocculated state, the mixing degree is lowered.
In the above described high-speed mixer, the powders are fluidized and mixed together in the flocculated or lumped stated so that the mixing degree is lowered.
In order to obviate this deficiency, there is proposed a mixer 90, known as the MGT mixer, in which, as shown in FIG. 10, cracking means 92 having multi-stage blades 93 adapted to be revolved at elevated speeds in a box-shaped casing 91 is separately provided at the lateral side of the casing in such a manner that the flocculated powders may be cracked simultaneously during mixing of the powdered to particulate materials.
In this mixer, since the cracking is performed simultaneously with mixing of the powdered to particulate materials, powders X an Y exist in the vicinity of the multi-stage blades of the crushing means, such that the flocculated powders X can not be cracked efficiently. Above all, with the higher mixing ratio of the powders Y, the powders X are less likely to impinge on the multi-stage blades and hence can be cracked only with considerable difficulties.
It becomes therefore necessary to crack the flocculated powders X before mixing the powders X and Y together.
On the other hand, when the powdered to particulate materials are mixed in the above described mixer, some materials undergo changes in the properties indigenous to them due to cracking in the course of mixing. Above all, the particulate materials, such as the granulated sugar having a particle size ranging between about 350 and 800 microns, are crushed by the agitating blades rotating at an elevated speed such that the properties indigenous to the materials are altered.
Although the number of revolutions of the agitating blades can be lowered to the extent that the particulated materials are not crushed in this manner, the mixing speed is then lowered undesirably.


A frequency synthesizer and method for synthesizing decimal frequencies. The synthesizer includes a seed generator, a clock synthesizer and an output synthesizer. The clock synthesizer includes a binary accumulator in a feedback signal path using a base reference frequency and a clock seed word for synthesizing a clock frequency and the output synthesizer includes a binary accumulator in a forward signal path using the clock frequency and an output seed word for synthesizing an output frequency. The output frequency is synthesized in decimal frequency steps from a decimal base reference frequency. Several output synthesizers may be driven in parallel from the same clock frequency for synthesizing several output frequencies in parallel.

1. Field of the Invention
This invention relates generally to signal frequency synthesis and more particularly to a frequency synthesizer and method for synthesizing exact multiples of decimal frequencies.
2. Description of the Background Art
Microwave frequency synthesizers play a crucial role in consumer, industrial and military applications. They are found in test systems for cell phones, radars and communication transceivers. The performance, cost and size of a synthesizer has a very substantial impact on commercial success and operational viability in numerous instances. Low phase noise, low spurious signal levels and fast switching speed are key performance factors.
Architectures for microwave frequency synthesis fit into the two broad categories of direct synthesis and indirect synthesis. The direct synthesis architectures use analog components such as mixers, filters, switches, frequency multipliers and frequency dividers for simple direct arithmetic frequency operations of addition, subtraction, multiplication and division. The resulting solutions have high performance, but when applied in the microwave frequency range, are costly, bulky and have high power consumption.
Indirect synthesis architectures make use of phase lock loops (PLL)s in the signal-switching-path. Single switching loop PLL architectures have low cost, small size and low power consumption by virtue of their simplicity. Such simple architectures are currently used in applications that do not require the level of performance demanded in military and high volume test applications. However, to meet a high level of performance, existing products use multiple phase-locked loops, sometimes as many as 8 or 10, in the signal switching path. As a result, the PLL implementations of high performance frequency synthesizers are also costly and bulky, and have high power consumption.
Both direct and indirect frequency synthesis use a reference frequency derived from a base reference oscillator for providing a highly stable, low phase noise signal from which all other frequencies are synthesized. While it is theoretically possible to use a base reference oscillator at any frequency, an oscillator having a decimal frequency of 10 MHz or 100 MHz is highly preferred because a great deal of engineering has gone into achieving high stability and low phase noise for the lowest cost for oscillators having these frequencies. Relatively low cost binary devices are used for fractional multiplication of the reference frequency for synthesizing the desired output frequency.
Frequency synthesis of exact multiples of 10 kHz, 100 kHz or 1 MHz is often desirable for communication and radar receiver test, and direct use as local oscillators in receivers and transmitters. These receivers and transmitters commonly use channel spacings that have exact decimal Hertz values (sums of multiples of 100 kHz). For example, GSM may have a channel spacing of exactly 200 kHz. Unfortunately, a 10 MHz or 100 MHz reference oscillator with fractional binary multiplication cannot directly provide steps sizes to exactly match these channel spacings. Of course, this problem can be resolved with ultra-fine resolution so that the frequency difference between the desired and actual step size is negligible. However, ultra-fine frequency resolution is expensive to achieve. The problem could be resolved using a binary base reference frequency, for example 225 at 33.554,432 MHz or 226 at 67.108864 MHz. However, base reference oscillators at these frequencies do not have all the benefits of existing base reference oscillators having decimal base frequencies of 10 MHz or 100 MHz.
There is a need for a low phase noise, low spurious signal level, fast switching microwave synthesizer at a low cost having frequency steps at exact multiples of a decimal frequency using a decimal frequency base reference frequency.