Limits of the distal inversion in the t complex of the house mouse: Evidence from linkage disequilibria

The suppression of crossing-over and the consequent linkage disequilibrium of genetic markers within the t complex of the house mouse is caused by two large and two short inversions. The inversions encompass a region that is some 15 centiMorgans (cM) long in the homologous wild-type chromosome. The limits of the proximal inversions are reasonably welldefined, those of the distal inversions much less so. We have recently obtained seven new DNA markers (D17Tu) which in wild-type chromosomes map into the region presumably involved in the distal inversions of the t chromosomes. To find out whether the corresponding loci do indeed reside within the inversions, we have determined their variability among 26 complete and 12 partial t haplotypes. In addition, we also tested the same collection of t haplotypes for their variability at five D17Leh, Hba-ps4, Pim-1, and Crya-1 loci. The results suggest that the distal end of the most distal inversion lies between the loci D17Leh467 and D17Tu26. The proximal end of the large distal inversion was mapped to the region between the D17Tu43 and Hba-ps4 loci, but this assignment is rather ambiguous. The loci Pim-1, Crya-1, and the H-2 complex, which have been mapped between the Hba-sp4 and Grr within the large distal inversion, behave as if they recombine from time to time with their wildtype homologs.     

Molecular characterization of four intra-t mouse recombinants

Recombination in the proximal region of mouse chromosome 17 is greatly reduced in heterozygotes carrying the wild-type and thet complex-type chromosomes. The reason for this is the presence of two non-overlapping inversions in thet complex. Rare crossing-over does, however, occur within thet complex of thet/+ heterozygotes. Here we characterize four such exceptional intra-t recombinants,t ^Tu1 throught ^Tu4 . To map the positions of the genetic exchange in these four recombinants, we analyzed them with DNA probes specific for 16 loci distributed over thet complex. The analysis revealed that in three of the four recombinants, an equal crossing-over occurred in the short region between the two inversions, producing chromosomes carrying either the proximal inversion only (t ^Tu1 andt ^Tu4 ) or the distal inversion only (t ^Tu2 ). In the fourth recombinant (t ^Tu3 ), unequal crossing-over occurred within the proximal inversion between lociD17Leh119 andD17Leh66, producing a chromosome in which the region containing lociTcp-1, T, andD17Tu5 has been duplicated. The duplication of theBrachyury locus leads to the suppression of the tail-shortening effect normally produced by the interaction of the dominant (T) and recessive (tct) alleles at this locus so that theT/t ^Tu3 mice have normal tails.     

INVERSION LENGTH AND BREAKPOINT DISTRIBUTION IN THE DROSOPHILA BUZZATII SPECIES COMPLEX: IS INVERSION LENGTH A SELECTED TRAIT?

Length and position of breakpoints are characteristics of inversions that can be precisely determined on the polytene chromosomes of Drosophila species, and they provide crucial information about the processes that govern the origin and evolution of inversions. Eighty-six paracentric inversions described in the Drosophila buzzatii species complex and 18 inversions induced by introgressive hybridization in D. buzzatii were analyzed. In contrast to previous studies, inversion length and breakpoint distribution have been considered simultaneously. We conclude that: (1) inversion length is a selected trait; rare inversions are predominantly small while evolutionarily successful inversions, polymorphic and fixed, are predominantly intermediate in length; a nearly continuous variation in length, from small to medium sized, is found between less and more successful inversions; (2) there exists a significant negative correlation between length and number of polymorphic inversions per species which explains 39% of the inversion length variance; (3) natural selection on inversion length seems the main factor determining the relative position of breakpoints along the chromosomes; (4) the distribution of breakpoints according to their band location is non-random, with chromosomal segments that accumulate up to eight breakpoints.     

Mapping of the Sod-2 locus into the t complex on mouse chromosome 17

A human DNA probe specific for the superoxide dismutase gene was used to identify the corresponding mouse gene. Under the chosen hybridizing conditions, the probe detected DNA fragments most likely carrying the mouse Sod-2 gene. Mapping studies revealed that the Sod-2 gene resides in the proximal inversion of the t complex on mouse chromosome 17. All complete t haplotypes tested showed restriction fragment length polymorphism which is distinct from that found in all wild-type chromosomes tested. The Sod-2 locus maps in the same region as some of the loci that influence segregation of t chromosomes in male gametes. The possibility that the Sod-2 locus is related to some of the t-complex distorter or responder loci is discussed. The data indicate that the human homolog of the mouse t complex has split into two regions, the distal region remaining on the p arm of human chromosome 6, while the proximal region has been transposed to the telomeric region of this chromosome's q arm.     

Sequence of the t complex Tcp-10a t gene and examination of the Tcp-10 t gene family

Transmission ratio distortion (TRD) is a property of complete t haplotypes which results in the preferential transmission of the t haplotype chromosome from heterozygous t/+ males to the majority of the offspring. A candidate gene for one of the primary genetic elements in TRD, the t complex responder locus has recently been suggested to be Tcp-10b ^t. There are multiple, functional Tcp-10 ^t genes, but genetic data suggest the presence of the Tcp-10a ^t gene alone is compatible with normal transmission ratios. Here we present the complete sequence and genomic structure of the Tcp-10a ^t gene which is compared with sequence data from a number of cDNAs and genomic subclones representing all active Tcp-10 ^t family genes. A detailed table of all sequence variants discovered in the course of our investigation is presented, and we have clarified the extent of 5 untranslated alternative splicing patterns exhibited by this gene family. A 60 base pair (bp) in-frame deletion from the 5 end of exon 3 of the Tcp-10a ^t gene is also presented and compared with the equivalent region of Tcp-10b ^t and Tcp-10c ^t. A search of the University of Edinburgh database has revealed a significant homology between the Tcp-10b ^t open reading frame and several cytosolic filament proteins. Interestingly, the region of homology is involved in the deletion from the Tcp-10a ^t gene.     

Chromosomal polymorphisms in the pitcher-plant mosquito,Wyeomyia smithii

A local population of the pitcher-plant mosquito, Wyeomyia smithii (Diptera: Culicidae, Culicini) in western New York State contains naturally polymorphic salivary gland chromosomes. Maps depicting the proposed standard sequence of bands along each of the three chromosomes are presented. Structural conformations of heterokaryotypes reveal ten paracentric inversions. Four inversions occur in chromosome 1, and three in each of chromosomes 2 and 3. All the inversions are small, occupying 3–17% of the length of their respective chromosomes, and all are located in the terminal 1/5 to 1/2 of a chromosome arm. Three inversions are almost adjacent on chromosome 1. On chromosome 2 one inversion lies completely within another. Three inversions on chromosome 3 partially overlap. The remaining two inversions occur alone, one on chromosome 1 and the other on chromosome 2. Another polymorphism, with no visible alteration of band sequence, produces asynapsis of all but the tip of the left arm of chromosome 1, and shows a significant change in frequency between two successive years. No inversion exhibits a significant seasonal change in frequency, though some fluctuation in frequency was noted in two cases. The rarest inversion in the population exists at a frequency below 0.01, and the two most common inversions at frequencies of 0.44 and 0.52. The average inversion heterozygosity of an individual is between 1.7 and 2.4 based on two different estimation procedures. Heterokaryotypes for several of the inversions were significantly more frequent than they should have been, given binomial expectations. Cytogenetic analysis of W. smithii was deemed particularly desirable because this species has been and continues to be the subject of extensive biometrical-genetic, ecological, and evolutionary investigation.This paper is the seventh in a series on the ecology and evolution of the pitcher-plant mosquito     

Nucleotide sequence of a mouse Tcp-1 pseudogene: A nucleotide record for a t complex gene carried by an ancestor of the mouse

We have isolated clones of a processed pseudogene of mouse t complex polypeptide 1 (Tcp-1) and determined the nucleotide sequence of the pseudogene. The pseudogene was 1363 bp long and had no intron. The Tcp-1 pseudogene had 88.4% or 88.3% nucleotide identity to the mouse Tcp-1 cDNA of wild-type (Tcp-1)^bor t haplotype (Tcp-1)^a, and 87.5% identity to the rat Tcp-1 cDNA. On 12 nucleotide positions where the open reading frames (ORFs) of mouse Tcp-1 ^band Tcp-1 ^acDNAs have bp substitutions, the Tcp-1 pseudogene had 6 bp identical to Tcp-1 ^b, 5 bp identical to Tcp-1 ^aand 1 bp not identical to neither. On ten amino acid positions where TCP-1B and TCP-1A polypeptides have substitutions, deduced amino acids of the Tcp-1 pseudogene had four amino acids identical to TCP-1B, five amino acids identical to TCP-1A and one amino acid identical to neither. These results suggest that the ancestral mouse Tcp-1 gene would have had no significant difference between the resemblance to Tcp-1 ^band that to Tcp-1 ^abefore they were diverged and that amino acids of TCP-1B and TCP-1A would have been substituted in similar high rates.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number D00851.     

Cytogenetic Features of Blackfly Wilhelmia paraequina Puri (Diptera: Simuliidae) from Armenia

Three blackfly Wilhelmia paraequina populations of Armenia (rivers Debet and Megriget, channel Megri) were studied. 2n = 6: IS + IIL, IIS + IL, IIIS + IIIL. Cytological maps of polytene chromosomes were constructed. High inversion polymorphism (95.63%) was observed, with 2.6 inversions per individual. Three inversions proved to be associated with male development: Y₁ was characterized by a combination of two heterozygous inversions, IIL-3 + IIL-1, while Y₂ had another combination of heterozygous inversions, IIL-5 + IIL-3. The X chromosome had a standard homozygous IIL band pattern. The populations were shown to be similar in autosomal polymorphism. A tendency for differentiation was observed with respect to frequencies and types of sex-linked inversions: the sex determination system was Y₁X–XX in the Debet and Megriget populations and Y₂X–XX in the channel (Megri) population. On the strength of these findings, one W. paraequina morphotype was assumed to involve two cytotypes, A (Debet and Megriget populations) and B (Megri population).     

A gene product of the mouse t complex with chemical properties of a cell surface-associated component of the extracellular matrix

p63/6.9 is a major testicular cell protein coded for by a gene, called Tcp-1, within the mouse t complex. All wild-type chromosomes carry the Tcp-1^b allele which codes for a basic form of this protein, while all complete mutant t haplotypes carry the Tcp-1^a allele which codes for an acidic form of this protein. Genetic studies have demonstrated a correlation between the Tcp-1 gene and certain t haplotype effects on sperm differentiation and maturation. In this report, an initial biochemical analysis of the p63/6.9 protein is presented. The data provide evidence that p63/6.9 is closely associated with the external surface of testicular cells but not as an integral membrane component. Some properties of the testicular form of this t complex gene product are similar to those reported for the matrix proteins fibronectin and laminin. The possibility is suggested that primary effects of t haplotypes on sperm differentiation could be exerted through the extracellular matrix. p63/6.9 is also present at a lower level within the cytoplasm and membranes of F9 teratocarcinoma cells. It appears that the level of p63/6.9 synthesis and the exact nature of p63/6.9 intra- and/or intermolecular interactions are under tissue-specific control.     

Further localization of ETS1 indicates that the chromosomal rearrangement in Ewing sarcoma does not occur at fra(11)(q23)

Summary A genomic probe homologous to 5.4 kb of the c-ets-1 gene was hybridized in situ to chromosomes expressing fra(11)(q23). This probe hybridized distal to the fragile site, which is just distal to the midpoint of band 11q23.3. This result localizes ETS1 from the FRA11B locus to 11q24. The result also distinguishes the FRA11B locus from the site of translocation at 11q23-q24 in the Ewing sarcoma- and peripheral neuroepithelioma-specific t(11;22), indicating that the chromosomes of a previously reported patient heterozygous for fra(11)(q23) did not rearrange at this fragile site to give rise to Ewing sarcoma. This adds to the mounting evidence against individuals with fragile sites being predisposed to developing cancer.     

Distribution of genetic diversity in relation to chromosomal inversions in the malaria mosquito Anopheles gambiae

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than threefold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.Correspondence to: K.D. Mathiopoulos     

Functional analysis of a t complex responder locus transgene in mice

Transmission ratio distortion (TRD) of mouse t haplotypes occurs through the interaction of multiple distorter loci with the t complex responder (Tcr) locus. Males heterozygous for a t haplotype will transmit the t-bearing chromosome to nearly all of their offspring. This process is mediated by the production of functionally inequivalent gametes: wildtype meiotic partners of t spermatozoa are rendered functionally inactive. The Tcr locus, which is required for TRD to occur, is thought to somehow protect its host spermatid from the sperm-inactivating effects of linked distorter genes (Lyon 1984). In previous work, Tcr was mapped to a small genetic interval in t haplotypes, and a candidate gene from this region was isolated (Tcp-10b ^t). In this work, we further localize Tcr to a 40-kb region that contains the 21-kb Tcp-10b ^t gene. A cloned genomic copy of Tcp-10b ^t was used to generate transgenic mice. The transgene was bred into a variety of genetic backgrounds to test for non-Mendelian segregation. Abberrant segregation was observed in some mice carrying either a complete t haplotype or a combination of certain partial t haplotypes. These observations, coupled with those of Snyder and colleagues (in this issue), provide genetic and functional evidence that the Tcp-10b ^t gene is Tcr. However, other genotypes that were predicted to produce distortion did not. The unexpected data from a variety of crosses in this work and those of our colleagues suggest that elements to the TRD system and the Tcr locus remain to be identified.     

Chromosomal evolution of the Hawaiian Telmatogeton (Chironomidae,Diptera)

It is only in the Hawaiian Islands that species of the otherwise marine genus Telmatogeton have evolved into freshwater. An analysis of polytene chromosomes and karyotypes of two marine species and five freshwater species revealed that paracentric inversions and centric fusions were important in chromosomal evolution. The sequence of polytene chromosome bands common to most species, established as the Telmatogeton standard sequence, is found in a population of T. torrenticola from West Maui. Most species and other populations of T. torrenticola may be derived from the standard sequence by paracentric inversions. Similarities with the standard band sequence places T. japonicus (n=7) rather than T. pacificus (n=4) in the proposed phylogeny as the species closest to the marine ancestor of the freshwater species. One of three species (T. fluviatilis from Oahu, T. torrenticola from West Maui, or an undescribed species from East Maui), each with seven pairs of chromosomes is considered to be closest to the original freshwater species. T. torrenticola is a complex species in which there is an accumulation of fixed inversions and centric fusions in stepwise fashion in populations from west to east (West Maui n=7; East Maui n=6; Kohala Mountains n=5 and Mauna Kea n=4 both from the island of Hawaii). The population of T. torrenticola from Molokai has a reduced chromosome number (n=4) and fixed inversions. T. abnormis and T. hirtus, the only species which exhibit differentiated sex chromosomes, may be derived from the standard sequency by paracentric inversions. T. abnormis (n=4) has a simple XY system and T. hirtus (n=3/4) has a complex XY₁Y₂ system. Unique sequences of bands, differences in staining intensity of puffs and bands, and an inversion form the basis for the differentiation of the various Y-chromosomes in these species.     

Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system

During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Therefore, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana. Via deep sequencing, we defined the patterns of junction formation in wild‐type and in the non‐homologous end‐joining (NHEJ) mutant ku70‐1. Surprisingly, in plants deficient of KU70, inversion induction is enhanced, indicating that KU70 is required for tethering the local broken ends together during repair. However, in contrast to wild‐type, most junctions are formed by microhomology‐mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg‐cell‐specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will.     

Mouse paracentric inversion In(3)55Rk mutates the urate oxidase gene

The paracentric inversion In(3)55Rk on mouse Chromosome 3 (Chr 3) was induced by cesium irradiation. Genetic crosses indicate the proximal breakpoint cosegregates with D3Mit324 and D3Mit92; the distal breakpoint cosegregates with D3Mit127, D3Mit160, and D3Mit200. Giemsa-banded chromosomes show the inversion spans approximately 80% of Chr 3. The proximal breakpoint occurs within band 3A2, not 3B as reported previously; the distal breakpoint occurs within band 3H3. Mice homozygous for the inversion exhibit nephropathy indicative of uricase deficiency. Southern blot analyses of urate oxidase, Uox, show two RFLPs of genomic mutant DNA: an EcoRI site between exons 4-8 and a BamHI site 3' to exon 6. Mutant cDNA fails to amplify downstream of base 844 at the 3' end of exon 7. FISH analysis of chromosomes from inversion heterozygotes, using a cosmid clone containing genomic wild-type DNA for Uox exons 2-4, shows that a 5' segment of the mutated Uox allele on the inverted chromosome has been transposed from the distal breakpoint region to the proximal breakpoint region. Clinical, histopathological, and Northern analyses indicate that our radiation-induced mutation, uox(In), is a putative null.     

STRUCTURE AND POPULATION GENETICS OF THE BREAKPOINTS OF A POLYMORPHIC INVERSION IN DROSOPHILA SUBOBSCURA

Drosophila subobscura is a paleartic species of the obscura group with a rich chromosomal polymorphism. To further our understanding on the origin of inversions and on how they regain variation, we have identified and sequenced the two breakpoints of a polymorphic inversion of D. subobscura—inversion 3 of the O chromosome—in a population sample. The breakpoints could be identified as two rather short fragments (～300 bp and 60 bp long) with no similarity to any known transposable element family or repetitive sequence. The presence of the ～300‐bp fragment at the two breakpoints of inverted chromosomes implies its duplication, an indication of the inversion origin via staggered double‐strand breaks. Present results and previous findings support that the mode of origin of inversions is neither related to the inversion age nor species‐group specific. The breakpoint regions do not consistently exhibit the lower level of variation within and stronger genetic differentiation between arrangements than more internal regions that would be expected, even in moderately small inversions, if gene conversion were greatly restricted at inversion breakpoints. Comparison of the proximal breakpoint region in species of the obscura group shows that this breakpoint lies in a small high‐turnover fragment within a long collinear region (～300 kb).     

Comparative polytene chromosome maps of <Emphasis Type="Italic">D. montana</Emphasis> and <Emphasis Type="Italic">D. virilis</Emphasis>

Chromosomal inversion polymorphism was characterized in Finnish Drosophila montana populations. A total of 14 polymorphic inversions were observed in Finnish D. montana of which nine had not been described before. The number of polymorphic inversions in each chromosome was not significantly different from that expected, assuming equal chance of occurrence in the euchromatic genome. There was, however, no correlation between the number of polymorphic inversions and that of fixed inversions in each chromosome. Therefore, a simple neutral model does not explain the evolutionary dynamics of inversions. Furthermore, in contrast to results obtained by others, no significant correlation was found between the two transposable elements (TEs) Penelope and Ulysses and inversion breakpoints in D. montana. This result suggests that these TEs were not involved in the creation of the polymorphic inversions seen in D. montana. A comparative analysis of D. montana and Drosophila virilis polytene chromosomes 4 and 5 was performed with D. virilis bacteriophage P1 clones, thus completing the comparative studies of the two species.     

Inversion polymorphism in populations of Drosophila sulphurigaster albostrigata and Drosophila nasuta albomicans from Phuket,Thailand

Two species of the Drosophila nasuta subgroup of the Drosophila immigrans group, D. sulphurigaster albostrigata and D. nasuta albomicans were investigated in this study. Collections of both species were made from Phuket, Thailand. Both species have similar salivary chromosomes, with four autosomal arms and one sex chromosome arm, and both are highly polymorphic for paracentric inversions. D. s. albostrigata accounted for the majority of the isolines collected and exhibited the greater number of inversions. One inversion, C₁, was common to both species, indicating common ancestry.A non-random distribution of inversions was observed on the proximal end of chromosome II in both D. s. albostrigata and D. n. albomicans. An inter-collection comparison revealed that both rigid and flexible chromosomal polymorphism were operating in the two species, with a seasonal variation noted for one inversion in D. s. albostrigata. A non-random association of two inversions was observed in D. n. albomicans.Based on a comparison of the indices of crossing over, both D. s. albostrigata and D. n. albomicans were found to be more heterozygous than in previous studies, with D. n. albomicans appearing to have evolved further than D. s. albostrigata.Based on a thesis submitted for the degree of Ph. D. at the University of Queensland.     

Polytene chromosome map and inversion polymorphism in Drosophila mediopunctata

Drosophila mediopunctata belongs to the tripunctata group, and is one of the commonest Drosophila species collected in some places in Brazil, especially in the winter. A standard map of the polytene chromosomes is presented. The breakpoints of the naturally occurring chromosomal rearrangements are marked on the map. The distribution of breaking points through the chromosomes of D. mediopunctata is apparently non-random. Chromosomes X, II and IV show inversion polymorphisms. Chromosome II is the most polymorphic, with 17 inversions, 8 inversions in the distal region and 9 in the proximal region. Chromosome X has four different gene arrangements, while chromosome IV has only two.     

Inversion polymorphism in the midge Glyptotendipes barbipes (Staeger)

Summary The salivary gland nuclei of larvalGlyptotendipes barbipes from Stratford (Ontario) comprise 3 pairs of long metacentric chromosomes (I–III) and one pair of short acrocentrics (IV). Homologues are closely paired when homozygous, centromeres are expressed as heterochromatic drums. Each chromosome carries a nucleolus, and Chromosome IV in addition carries a Ring of Balbiani. The gross polytene idiogram is identical with that implicit inBauer's cytological analysis of the same species from Germany.In direct comparison the banding pattern of the German and Canadian larvae proved identical for at least one sequence in each chromosome arm.Three simple inversions are known in the species; one (III L-1) is endemic to Germany, a second (II L-3) has been found only once in Canada and a third (I S-1) is common in Canada but has not been reported from Germany. The remaining 2 inversions (II L-1,2 and III S-1,2) are complex and are here analysed as included types. Rearrangement II L-1,2 occurs both in Canada and in Germany and achieves heterozygous frequencies near 50% in both countries. It is the first inversion reported to be holarctic in distribution in insects not associated with man. The second complex inversion III S-1,2 has been found only in Canada where it is common. In each of the complex inversions at least one pair of breaks is near coincident.Meiosis is normal and chiasmatic in structurally homozygous males. It lacks conspicuous localization of chiasmata. No meiotic abnormalities were observed in inversion heterozygotes. It is postulated that complex heterozygous inversions interfere with pairing to such an extent that the residual ill-effects are out balanced by heterosis of co-adapted systems.A selective advantage of heterozygosis is directly indicated for II L by a significant (p=0.02) excess of II L/II L-1,2 larvae over the Hardy-Weinberg expectation.