Narrowing the Critical Regions for Mouse t Complex Transmission Ratio Distortion Factors by Use of Deletions

Corresponding author: Mary F. Lyon, MRC Mammalian Genetics Unit, Harwell, Didcot, Oxon OX11 0RD, United Kingdom., m.lyon{at}har.mrc.ac.uk (E-mail)

Communicating editor: N. A. JENKINS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Previously a deletion in mouse chromosome 17, T^22H, was shown to behave like a t allele of the t complex distorter gene Tcd1, and this was attributed to deletion of this locus. Seven further deletions are studied here, with the aim of narrowing the critical region in which Tcd1 must lie. One deletion, T^30H, together with three others, T^31H, T^33H, and T^36H, which extended more proximally, caused male sterility when heterozygous with a complete t haplotype and also enhanced transmission ratio of the partial t haplotype t⁶, and this was attributed to deletion of the Tcd1 locus. The deletions T^29H, T^32H, and T^34H that extended less proximally than T^30H permitted male fertility when opposite a complete t haplotype. These results enabled narrowing of the critical interval for Tcd1 to between the markers D17Mit164 and D17Leh48. In addition, T^29H and T^32H enhanced the transmission ratio of t⁶, but significantly less so than T^30H. T^34H had no effect on transmission ratio. These results could be explained by a new distorter located between the breakpoints of T^29H and T^34H (between T and D17Leh66E). It is suggested that the original distorter Tcd1 in fact consists of two loci: Tcd1a, lying between D17Mit164 and D17Leh48, and Tcd1b, lying between T and D17Leh66E.

TWO of the peculiar features of the t complex on mouse chromosome 17 are the abnormally high transmission of the t-carrying chromosome to the offspring of male mice heterozygous for a complete t haplotype and the sterility of males homozygous for a nonlethal t haplotype or doubly heterozygous for two different lethal haplotypes. These phenomena have been attributed to the action of distorter genes on a responder gene in the complex. There are thought to be at least three distorters, Tcd1, Tcd2, and Tcd3, at different points in the complex, which act additively to produce a harmful effect on the wild-type allele of the responder (LYON 1984 , LYON 1986 , LYON 1987 ). The t form of the responder, Tcr^t, is relatively resistant to this harmful action and hence, in heterozygotes, sperm carrying Tcr^t preferentially take part in fertilization, leading to high transmission of the t haplotype. When the distorters are homozygous their deleterious effects are so severe that the resistance of Tcr^t is overcome and the harmful effects on both Tcr⁺ and Tcr^t mean that no sperm can fertilize and the male is sterile. Thus, the distorters are thought to be responsible for both transmission ratio distortion (TRD) and male sterility. The t complex occupies the proximal third of chromosome 17 (LYON et al. 1986 , LYON et al. 1988 ) and is maintained intact by four nonoverlapping inversions that prevent crossing over (HAMMER et al. 1989 ) (Fig 1). The responder locus lies near the center of the complex and the distorter loci are spread through the region with Tcd1 proximally, Tcd2 in the distal inversion, and Tcd3 just distal to the responder. The responder gene has recently been identified (HERRMANN et al. 1999 ), but none of the distorters has so far been cloned, although various candidate genes have been suggested.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Diagram of structure of the t complex. The four inversions are shown with the wild-type (wt) orientation above and t complex (t) below, and the centromere to the left. The relative positions of some known genes and DNA markers are indicated, as are the approximate positions of the t complex distorters Tcd1 to Tcd3 and responder Tcr. The numbered symbols are abbreviations for DNA markers, full symbols, D17Leh48, etc.

Previously, we studied a deletion, T^22H, which covers the locus of brachyury, T, and also the Tcd1 locus (LYON 1992 ). It behaved like the t allele of Tcd1, causing male sterility in heterozygotes with a complete t haplotype, t^w32, and enhanced TRD when heterozygous with a partial t haplotype carrying the responder. In the present article the effects on male sterility and TRD of seven further deletions covering the T locus have been studied. The aim was to narrow the critical region for the Tcd1 locus and thus aid in identification of the underlying gene. The deletions arose in radiation mutagenesis experiments or spontaneously. Some of these deletions have already been used in positioning the head-tilt (het) locus that lies proximal to T (BERGSTROM et al. 1998 ). The results complement those of PLANCHART et al. 2000 . The region in which Tcd1 must lie is considerably reduced, and the possibility that it consists of the two separable loci Tcd1a and Tcd1b is put forward.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Origins:
All except one of the deletions studied were induced by radiation treatment of males (Table 1), in some cases combined with chemical treatment. In the case of T^29H the treated male was from the BALB/c strain, and the single spontaneous deletion T^34H was found in a mixed mutant stock. In all other cases the treated male was of the F₁ hybrid stock C3H/HeH x 101/H (3H1).

Breeding:
Where possible the deletions were maintained by crossing to tufted animals from the inbred strain TFH/H, tufted tf, being a recessive mutant causing hair loss located a few centimorgans from the T locus, and the TFH/H strain being genetically T tf/+tf x +tf/+tf. However, in some cases these crosses bred poorly, and the deletions concerned were then maintained by crosses to 3H1.

The t haplotypes used were t⁶, t^h2, t^h51, and t^w32, carrying the ratio and sterility factors shown in Table 2. They were maintained in separate closed stocks on nonstandard backgrounds.

Fertility tests:
Males to be tested for fertility were placed with two +tf/+tf females from the TFH/H strain and left for 1 mo. If neither female became pregnant the male was judged to be sterile and the females were killed. If young were produced the mice were left to breed for at least 3 mo and the number of young per female per month was calculated as a measure of the male's fertility.

Transmission ratio:
For tests of transmission ratio Tⁿ/t males were crossed to +tf/+tf females and numbers of short-tailed (Tⁿ/+) and normal-tailed (t/+) young were counted at birth. The control t/+ male sibs or half-sibs were crossed to Ttf/+tf females and numbers of short-tailed (T/+) and tailless (T/t) as well as normal-tailed (t/+ or +/+) young were counted.

Molecular mapping:
For mapping the positions of the breakpoints of the deletions, some mice of genotype Tⁿ/t^w32 were studied to make use of polymorphisms associated with the t complex. In other cases the deletions were crossed with Mus castaneus. Restriction fragment length polymorphisms and simple sequence variants were used as in BERGSTROM et al. 1998 .

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Characterization of the deletions:
(a) Extent of deletions: All the deletions were originally detected through their short-tailed phenotype in heterozygotes. Allelism with T was shown by a tailless phenotype in offspring of crosses with the t haplotype t^h2 and by linkage with tufted, tf. Evidence for the presence of deletions was provided by crosses to two nearby loci, quaking (qk), and head-tilt (het) (see Fig 3) and by cytogenetic tests. All except T^30H gave the quaking phenotype when crossed with qk/qk, indicating deletion of the qk locus, distal to T (Table 3). Three deletions, T^31H, T^33H, and T^36H, gave the head-tilt phenotype when crossed with het/het (Table 3), indicating deletion of the het locus, which lies proximal to T (BERGSTROM et al. 1998 ). To test for deletion of the T-maternal effect (Tme) locus, deletions were tested for transmission from the female. Only T^34H/+ was not viably transmitted from females. Very few T^34H/+ offspring of T^34H/+ females survived birth, and in dissections of pregnant T^34H/+ females the short-tailed offspring showed the edema typical of fetuses with the Tme phenotype (data not shown).

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Cytogenetic evidence of deletions. Some representative examples are shown, in each case with the deleted chromosome on the right. A shortening of the 17A3 band can be seen (uppermost light band).

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Diagrammatic representation of the extent of the various deletions. Solid lines indicate regions deleted, with dotted extensions showing uncertainty. Symbols of some markers are abbreviated: T48, 66E, and 119 by removal of D17Leh; Tu1 and Aus9 by removal of D17; and others (except het, T, qk, and Tme) by removal of D17Mit. Distances are not to scale. The critical regions for location of Tcd1a and Tcd1b are shown.

The cytogenetic tests provided further evidence of deletions. All the deletions showed visible shortening of the band 17A3, this being most marked in T^33H (Fig 2). With T^30H, the cytogenetics provided the only evidence at this stage of a deletion.

Since all these mutations involve cytogenetically visible deletions, they should be given the appropriate genetic symbols. These are Del(17)T^29H84H, etc. (Table 1). For convenience these symbols will be here abbreviated as T^29H, etc.

(b) Viability and fertility: It was necessary to know the viability and fertility of heterozygotes for the deletions with wild type before assessing effects on them of t complex factors. Tests revealed deleterious effects of the three deletions, T^31H, T^33H, and T^36H, which extended to cover het on both viability and fertility. Owing to poor breeding in crosses to the inbred strain TFH/H, it was necessary to maintain these deletions by crosses to the F₁ hybrid 3H1. All three then showed a statistically significant shortage of short-tailed offspring (Table 4). This was particularly marked in the case of T^36H, where only 27.1% of young born were short tailed. Although female T^36H/+ bred very poorly, the proportion of short-tailed young among their offspring was very similar to that from males, and thus T^36H did not show a Tme phenotype. Other possible causes of the deficiency of affected offspring include incomplete penetrance, reduced viability, or some form of distorted transmission. The evidence pointed to reduced viability. If incomplete penetrance were the case, then in crosses with qk or het, some normal-tailed quaking or head-tilt young might be found, and this was not so. In addition the shortage of affected young was still evident when T^36H was crossed to t^h2/t^h2 or t^h51/t^h51 when, owing to the obvious tailless phenotype, incomplete penetrance would not be expected. Further, among offspring of T^36H+/t^h2tf or T^36H+/t^h51tf, incomplete penetrance might lead to an excess of apparent crossovers of the type normal-tailed nontufted and again this was not so. Dissections of pregnant females failed to reveal the cause of the excess death of T³⁶/+ young. The deletions T^31H and T^33H also showed mild shortages of short-tailed offspring. Again there was no evidence of incomplete penetrance, and the shortages were ascribed to reduced viability. T^29H also showed mildly reduced viability.

The three deletions that covered the het locus also showed impaired fertility. With the four deletions that did not cover het, all Tⁿ/+ males crossed to non-T females proved fertile (Table 5). For T^31H, T^33H, and T^36H, however, some Tⁿ/+ males were sterile. The underlying basis of the impaired viability and fertility of these deletions is not known. It could be an effect of t complex factors but the deletions probably extend beyond the proximal limit of the t complex and hence other factors may be involved. In any case these impairments need to be taken into account in studies of the fertility and TRD of males also carrying t haplotypes.

Effect of deletions on t-complex male sterility:
The fertility of males carrying the partial t haplotypes t^h2 and t^h51 opposite a deletion was compared with that of similar males carrying the complete t haplotype t^w32 (Table 5). The t^h2 and t^h51 haplotypes were used as controls. Male sterility due to the t complex typically occurs when at least one t complex distorter gene is homozygous. t^h2 carries no distorters and thus, even if the deletion behaved like the distorter Tcd1^t, males of genotype Tⁿ/t^h2 would be expected to be fertile. t^h51 carries Tcd1^t but no other distorters. In earlier work, homozygosity for Tcd1^t alone did not result in male sterility, and thus Tⁿ/t^h51 would be expected to be fertile. By contrast, males of genotype t^h51/t^w32 are typically sterile due to homozygosity of Tcd1^t combined with heterozygosity of Tcd2^t and Tcd3^t. Similarly, T^22H/t^w32 males are sterile, presumably due to the deletion of the Tcd1 locus in T^22H. Sterility of Tⁿ/t^w32 males carrying the new deletions would thus indicate deletion of the Tcd1 locus.

As expected, all males of the control genotypes Tⁿ/t^h2 were fertile, consistent with the absence of any distorter genes in t^h2. With t^h51, however, the results were somewhat unexpected. All males carrying the deletions not covering het were fertile. However, several males carrying the deletions T^31H and T^33H were sterile, and the remainder sired a low number of young per female per month. Although all of four T^36H+/t^h51tf males were fertile, again the number of young sired was abnormally low. Here the sterility of some heterozygotes with wild type has to be taken into account. Other evidence given below suggests that T^31H, T^33H, and T^36H all delete Tcd1. Thus, the heterozygotes with t^h51 would in effect be homozygous for Tcd1^t, and a relatively mild impairment due to this may be acting additively with factors causing sterility of some heterozygotes with wild type.

When tested against t^w32, the deletions T^29H, T^32H, and T^34H gave fully fertile males (except for one T^29H+/t^w32+ male). By contrast, all of six T^30H/t^w32 males were completely sterile. This is similar to the results previously obtained with T^22H and is consistent with the T^30H deletion covering the Tcd1 locus. The T^30H/t^w32 males would thus have no normal copies of Tcd1 and would also carry t alleles of the remaining distorters.

Among the deletions covering the het locus, all of three T^31H/t^w32 males were sterile. Out of eight T^33H/t^w32 males, seven were fertile, but all were poorly fertile, with an average of only 1.1 young per female per month, well below the normal value. With T^36H, the heterozygotes with t^w32 were poorly viable and no males survived to adulthood. If the poor fertility of T^33H/t^w32 males is considered as a variant of the sterility due to t complex factors, these results with T^31H and T^33H are consistent with deletion of Tcd1. As these deletions cover the het locus they must extend more proximally than T^30H, and hence their deletion of Tcd1 is as expected.

Effects of deletions on transmission ratio distortion:
Data on TRD were obtained with the t haplotypes t⁶, t^h2, t^h51, and t^w32, but the main test was with t⁶. t^h51 served as a negative control, since it does not carry the responder Tcr^t. For TRD to occur the responder must be heterozygous. Therefore, no TRD is expected among the offspring of Tⁿ/t^h51 males. The results were as expected except for T^32H/t^h51tf, which gave a shortage of t^h51 offspring (Table 6). The explanation of this is not clear, but since the responder Tcr^t is not present in these males there is no reason to suppose that this discrepancy is due to t complex factors. With T^36H/t^h51 there was a shortage of T^36H/+ offspring but this is as expected from the poor viability of T^36H/+. The t^w32 haplotype was a positive control. Since it is a complete haplotype, Tⁿ/t^w32 males would be expected to give strong TRD in favor of t^w32 with all Tⁿ that permitted fertility of such heterozygotes, but the TRD might be so high that any enhancement by the deletions could not be detected. This was indeed found. An unexpected result was that of heterogeneity among the deletions, with T^34H giving a lower TRD of t^w32 than the other three. Of the four deletions involved, T^33H is thought to delete the Tcd1 locus (see above) whereas the other three apparently do not. Thus, these three, T^29H, T^32H, and T^34H, might have been expected to give similar TRD of t^w32. However, the heterogeneity ² among this group is 27.82, with 2 d.f. and P < 0.0001.

The t^h2 and t⁶ haplotypes were the test haplotypes. t^h2 carries the responder but no distorters, and in heterozygotes with wild type it is transmitted in a low ratio. The deletion T^22H, in which the distorter Tcd1 is deleted, raised the ratio of t^h2 in T^22H/t^h2 heterozygotes (LYON 1992 ). In the present work, all deletions gave very similar ratios, with the exception of T^36H. The proportion of t^h2 offspring of T^36H/t^h2 males was 46.7%. However, the poor viability of T^36H/+ young mentioned above must be taken into account, since it alone could give an apparent increase in the ratio of t^h2 young born. If the viability of T^36H/+ relative to +/+ or +/t is , and the true transmission ratio of the t haplotype concerned is r, then among the offspring of T^36H/t males the ratio of t carrying to total offspring scored at birth is given by

where t and N indicate the numbers of t and total offspring, respectively.

From the data on viability of T^36H given earlier, 27.1% of T^36H/+ offspring were found when, with full viability, 50% would be expected. Therefore, the viability is given by

and

Therefore, if t and N are 63 and 135, then

Thus, when allowance is made for the reduced viability of T^36H/+ young the transmission ratio of T^36H/t^h2 males is very similar to that of the other deletions (Table 6). The data in Table 4 showed that three other deletions, T^29H, T^31H, and T^33H, also had slightly reduced viability. Correction of the transmission ratios of these for viability produced only small changes: 20.7, 26.8, and 23.8%, respectively. This means that the data provide no evidence that any of this group of deletions alters the TRD of t^h2. In view of the clear enhancement of TRD found earlier with T^22H the reason for the present negative finding is not clear.

The main test of ratio distortion was made with t⁶, since its mildly raised ratio in T/t⁶ or +/t⁶ males provides scope for detection of changes. Because of the known dependence of TRD on genetic background, test males of genotype Tⁿ/t⁶ were compared with their sibs or half-sibs of genotype +/t⁶. Where possible, the test males and sibs were bred without outcrossing the stocks. However, in some cases the poor breeding behavior of the deletions necessitated the use of animals derived from outcrosses, and the data from outcross and nonoutcross animals are shown separately.

(a) Deletions not covering the het locus: For these deletions (T^29H, T^30H, T^32H, and T^34H) almost all the data were from non-outcross animals. The four sets of data from the control t⁶+/+tf sibs showed good agreement (heterogeneity ² = 1.28, d.f. = 3, P = 0.734), with transmission ratios ranging from 46.3 to 61.1% (Table 7). This indicates that the general genetic backgrounds of the stocks were reasonably similar. By contrast, among the sets of data from Tⁿ+/t⁶+ males there was strong heterogeneity (² = 107.42, d.f. = 3, P < 0.0001), T^30H giving a markedly high value of 95.8% and T^34H a lower value of 51.9%. When T^30H was removed from the test, heterogeneity still remained (² = 58.7, d.f. = 2, P < 0.0001), but when T^30H and T^34H were both removed, the two remaining deletions T^29H and T^32H showed good agreement (² = 0.122, d.f. = 1, P = 0.728). Of the four deletions only T^34H showed no significant difference from its control. T^29H and T^30H both showed a highly significant difference. T^32H, with fewer data, gave a marginally significant ², but some data were also available from outcross animals, and again a significantly raised transmission of t⁶ from the test males was seen (Fisher's exact test P < 0.0001). Correction of the T^29H data for reduced viability of T^29H produced only a small change in ratio from 84.9 to 79.7% and did not affect the conclusion.

Thus, it appeared that T^29H, T^30H, and T^32H all led to an increased TRD of t⁶, with T^30H having a significantly stronger effect than the other two.

(b) Deletions covering the het locus: For the deletions T^31H, T^33H, and T^36H only data from outcross animals were available. The outcrosses led to an increase in the TRD of the control t⁶/+ males, making detection of any enhancing effect of the deletions more difficult (Table 7). There was statistically significant heterogeneity among the transmission ratios of the three sets of control t⁶/+ males (² = 26.05, d.f. = 2, P < 0.0001), but none among the data from the test males (² = 4.25, d.f. = 2, P = 0.120). All three groups of test males gave a very high transmission ratio, ranging from 96.6 to 99.0%. With T^33H and T^36H there was a significant difference between test and control males, but with T^31H there was no such difference. The TRD of control t⁶/+ males for T^31H was very high, at 97.3%. For this reason any enhancing effect of T^31H would be very difficult to detect, and hence the interpretation of the negative result with T^31H is not clear.

As in the tests with t^h2, it is necessary to correct for the reduced viability of T^36H/+. As before the viability, , of T^36H/+ is taken as 0.37. Using the same formula as before

Thus, after allowing for the reduced viability, the TRD of T^36H/t⁶ is still considerably higher than that of the t⁶/+ sibs. Correction for the mildly reduced viability of T^31H and T^33H resulted in only small changes, to 96.1 and 98.7%, and did not affect the conclusions.

Molecular mapping of deletion breakpoints:
Some idea of the extent of the deletions had already been obtained by crosses to qk and het and by cytogenetic tests. To obtain more precise estimates of the positions of the deletion breakpoints mice heterozygous for the deletions and a t haplotype or with M. castaneus were analyzed for informative polymorphisms. Some of the results have already been published (BERGSTROM et al. 1998 ). Further work showed that in T^30H the markers D17Mit196 and D17Tu1 were deleted (Table 8 and Fig 3), whereas in T^29H and T^32H they were not. In addition, in T^32H the locus of T48 was not deleted. Because T^30H shows the phenotype of a deletion of Tcd1, whereas T^29H and T^32H do not, these mapping results imply that the Tcd1 locus is situated between the markers D17Leh48 and D17Mit164.

The proximal breakpoint of T^34H lay more distally, since this deletion did not extend to the marker nearest to the brachyury locus, D17Leh66E. T^29H and T^30H both deleted the D17Leh66E locus. Phenotypically T^34H differed from T^29H and T^32H in that the latter two enhanced the TRD of t⁶ and T^34H did not. It is possible that this difference is attributable to deletion in T^29H and T^32H of a distorter locus lying between T and D17Leh66E.

Among the distal breakpoints, that of T^30H was the most proximal and that of T^34H the most distal (Table 3 and Fig 3). Thus, there is no evidence that the phenotypic differences among the deletions could be attributed to the positions of their distal breakpoints or to an unspecific effect of their general length.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The deletions studied here extend knowledge of the genetic basis of TRD due to the t complex and of the identity and location of distorter and male-sterility factors in the proximal region of mouse chromosome 17. Previously (LYON 1992 ) we had shown that the deletion T^22H behaved as though it deleted the locus of Tcd1 and that the effect of this deletion was like that of a t allele at this locus. The aim of the present work was to find which of the new group of deletions behaved like T^22H, and thus to narrow the critical region for Tcd1.

Concerning male sterility the effect of the T^30H deletion was very clear. Heterozygotes of T^30H with wild type or with the two partial haplotypes t^h2 and t^h51 showed normal fertility, but heterozygotes with the complete t haplotype t^w32 were totally sterile. In this, T^30H resembles T^22H. The deletions T^31H and T^33H also impaired male fertility but the effects were less clear. In the case of T^33H, T^33H/t^w32 males showed much impaired fertility, rather than total sterility. With T^31H, the sterility of T^31H/t^w32 males was total, but some T^31H/+ males were also sterile, complicating the interpretation. With T^36H, no T^36H/t^w32 males could be tested. T^31H, T^33H, and T^36H were all shown to delete the locus of het, which lies proximal to T (BERGSTROM et al. 1998 ), and hence to extend more proximally than the rest (Fig 3). Thus, these results are consistent with T^30H, T^31H, T^33H, and T^36H all deleting the locus of a t complex sterility factor, presumably Tcd1, and, as with T^22H, absence of this locus having an effect like a t allele.

The current interpretation of the effects of the t complex on TRD and male sterility is that the sterility is due to homozygosity of distorter genes that, when heterozygous, result in TRD. The deletions that affected male sterility would therefore be expected also to affect TRD. As expected from its effect on male sterility, T^30H showed a strong enhancement of TRD of t⁶. T^33H and T^36H also showed ratio distorting effects, as expected. With T^31H no significant effect on TRD was detected. Nevertheless, the transmission of t⁶ from the test males was very high and the failure to detect any enhancement of ratio could be due to the unusually high TRD of the control males in this test. Thus, the results concerning TRD are in accord with the effects on male sterility.

These results narrow the critical region in which the Tcd1 locus lies. Previously, the results with T^22H had placed this locus between a point proximal to the D17Tu1 locus and the locus of brachyury (HOWARD et al. 1990 ). Among the present group of deletions, the breakpoint in T^30H is distal to D17Mit164 and in T^32H is proximal to D17Leh119, placing the Tcd1 locus between these two markers (Fig 3). The data of PLANCHART et al. 2000 on embryonic stem cell-derived deletions narrow the interval further to between D17Aus9 and Tctex1. Thus, the interval in which this locus can lie is now quite small, and this knowledge will be valuable in cloning the gene. The critical region now appears to exclude the locus of Tctex1, previously a possible candidate gene (LADER et al. 1989 ; HOWARD et al. 1990 ; O'NEILL and ARTZT 1995 ).

An unexpected result was an effect of the deletions T^29H and T^32H in enhancing TRD, but not inducing male sterility. In each case the effect was highly significant, but also significantly lower than that seen with T^30H. This raises the possibility of deletion of another distorter locus in T^29H and T^32H but not in T^34H. T^34H showed no effect on TRD although it extends distally farther than all the other deletions studied here in that it deletes the Tme locus. Thus, this second effect on TRD is not likely to be due to a locus distal to T or to an unspecific effect of the length of the deletions. However, proximally T^34H extends for the least distance. It fails to delete the locus of D17Leh66E, whereas T^29H and T^32H both delete this locus. Thus, it is possible that there is a distorter locus situated between T and D17Leh66E (not deleted in T^34H; Fig 3). Then T^34H would have no distorter loci deleted, T^29H and T^32H would have one, and the remaining deletions, T^30H, T^31H, T^33H, T^36H, and T^22H, studied previously, would have two. It is of interest that T^34H also gave a lower transmission of the complete haplotype t^w32 than did T^29H and T^32H.

If there is indeed a distorter locus between T and D17Leh66E, then the hairpin-tail deletion, T^hp, should also show an enhancing effect on TRD since, like T^29H, it deletes D17Leh66E (BERGSTROM et al. 1998 ). It is not possible to test males of genotype T^hp/t⁶, since T^hp carries the t⁶ recessive lethal factor. However, in earlier work T^hp enhanced the transmission of t^h2 from T^hp/t^h2 males (LYON 1992 ).

Deletion of this apparent new distorter did not result in sterility of males also carrying the complete haplotype t^w32. This raises the question whether the current interpretation that homozygosity of distorters leads to sterility is correct, or whether there are distorters that do not affect fertility. It seems not possible to say. The effect of the various distorters is cumulative, both on TRD and on sterility. It is known that homozygosity for the partial haplotype t^h51, previously thought to carry Tcd1 and now also appearing to carry the new distorter, does not lead to sterility. Perhaps the sterility seen in T^30H/t^w32 (and also in t^h51/t^w32) requires homozygosity or deletion of both Tcd1 and the new distorter (as well as the presence of Tcd2^t and Tcd3^t). This distorter may impair sperm function but not sufficiently to cause sterility when it is the only homozygous locus. On the other hand, it is also possible that there are two types of distorters, some affecting fertility and others not. Among this group of deletions there is no evidence for a sterility locus not affecting ratio distortion.

The suggestion of this new distorter implies that there are more distorters than the three (Tcd1, Tcd2, and Tcd3) originally postulated. There have already been such suggestions from other work. SILVER and REMIS 1987 postulated the existence of Tcd4, located distal to T, in the region of Tcp1, and SILVER 1989 suggested a Tcd5 locus lying distal to Tcd3. From their positions neither of these could be identical with the new distorter described here. GUMMERE et al. 1986 studied the effect of genetic background on TRD and found influences of the general genetic background and of the homologous chromosome 17. An effect of chromosome 17 could be due to polymorphisms among wild-type alleles of distorter genes, but Gummere et al.'s work did not identify the location of the genes concerned. A problem with the present work is that effects of genetic background cannot be excluded. T^29H, T^30H, T^32H, and T^34H were all maintained by crossing to the inbred strain TFH/H and had at least two to three backcrosses to this strain when the crosses to t⁶ were made. Hence, their general genetic backgrounds were reasonably similar, and this is confirmed by the homogeneity of the four sets of control males from these crosses. However, these deletions arose by mutation in different strains. T^29H arose in BALB/c and T^34H in a mutant stock, whereas T^30H and T^32H arose in the F₁ strain 3H1 (C3H/HeH x 101/H). Thus, it is possible that there could be polymorphism in undeleted regions of chromosome 17 that are responsible for the effects. The identification of the new distorter is therefore tentative. However, support for its existence is provided by the work of BENNETT and ARTZT 1990 who found that the three deletions T^Or, T^hp, and T^Orl, all with proximal breakpoints between the markers D17Leh48 and D17Leh66E, also enhanced TRD but did not cause male sterility when opposite a complete t haplotype. This means that all five deletions (these three and T^29H and T^32H) that delete the region between T and D17Leh66E have a similar effect on TRD.

Another difficulty in the identification of distorters is the crossover suppression due to the four inversions in the t complex. The three original distorters were identified by study of partial t haplotypes arising by crossing over. However, this crossing over leaves chromosomal segments still intact. The Tcd1 locus was identified by absence of its t allele from the t⁶ haplotype. This haplotype has wild-type chromatin in the proximal region of chromosome 17 extending distally as far as D17Aus3II (HOWARD et al. 1990 ). Since the exact position of the new distorter is not known it is not clear whether t⁶ has a t or wild-type allele of this distorter. The results appear to suggest that it has a wild-type allele. If so, the effect of the present results is to indicate that Tcd1 can be split into two loci, one deleted in T^29H and T^32H and both deleted in T^22H and T^30H. Provisionally, the more proximal one may be designated Tcd1a and the other Tcd1b.

The recent identification of the responder as a mutant form of a sperm motility kinase, Smok (HERRMANN et al. 1999 ), makes it possible to speculate on the function of the distorters. HERRMANN et al. 1999 suggest that they may constitute components of a signal cascade regulating Smok functions, which in turn may affect sperm flagellar function. This knowledge, combined with the narrowing of the critical region for Tcd1 provided by the present work, should bring closer the cloning of the distorters and hence a full understanding of TRD by the t complex.

	ACKNOWLEDGMENTS

We are grateful to Bruce Cattanach for the gift of T^29H–T^33H and T^36H and to Colin Beechey for the gift of T^34H. We thank Mark Harrison for animal care. M.F.L. was partly supported by EU contract no. CHRX-CT93-0181. The animal stocks at Harwell were maintained under the guidance issued by the Medical Research Council in "Responsibility in the Use of Animals for Medical Research" (July 1993) and Home Office Project Licence no. 30/00875.

Manuscript received November 19, 1999; Accepted for publication February 28, 2000.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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