Genetic analysis of the H-2D region using a new intra-D-region recombinant mouse strain

A rare D-region recombination event which gave rise to the B10.RQDB major histocompatibility complex haplotype has been examined to ascertain the nature of the crossover and to determine which class I genes are present in the new alignment of D-region genes. Serologic analysis have shown that the B10 . RQDB major histocompatibility complex recombinant mouse inherited the H-2Dd gene from the B10.T(6R) parental line and the H-2Db gene from the B10.A(2R) parental line, representing the first example of an intra-D-region crossover resulting from an intercross. Previous molecular genetic analyses of the d and b haplotypes revealed structural diversity in the organization of their D-region gene clusters. Hence, the D region is comprised of five class I genes in the d haplotype and only one in the b haplotype. Because allelic relationships among the various D-region genes are not defined, either a homologous or nonhomologous alignment of genes has generated the RQDB crossover. Therefore, the possibility that all three D-region antigen-presenting molecules (Dd, Ld, and Db) might be encoded by the RQDB haplotype was examined. Fluorescence-activated cell sorter and cytotoxic T lymphocyte analyses revealed no detectable levels of H-2Ld cell-surface expression, confirming earlier studies with antibody-mediated cytotoxicity and immunoprecipitation. Southern blot analysis localized the recombination point to within a 1-kb region at the centromeric end of the H-2Ld gene on the B10 . T(6R) chromosome in a region of high homology to the H-2Db gene on the B10 . A(2R) chromosome. Together, these studies define the D region of the RQDB haplotype as containing the five class I genes: Dd, D2d, D3d, D4d, and Db. In addition to providing insight into rare recombination events in the D region, the B10.RQDB mouse should be a useful tool for exploring the function of D-region genes.     

QUOD ERAT FACIENDUM: sequence analysis of the H2-D and H2-Q regions of 129/SvJ mice

The H2-D and -Q regions of the mouse major histocompatibility complex ( Mhc or H2) have been sequenced from strain 129/SvJ (haplotype bc), revealing a D/Q region different from all other investigated haplotypes, including the closely related b haplotype. The 300-kb class I-rich region consists of the classical class I, H2-D, and 11 non-classical class I genes. The Q region was formed by two series of tandem duplications. Comparison of the segment between the D and Q1 genes with the H2-K region provides evidence that class I genes were translocated from the K region to the D region, and gives a new explanation for the weak locus specificity of the H-Kand H2-Dalleles.     

Comparative anatomy of the primate major histocompatibility complex DR subregion: evidence for combinations of DRB genes conserved across species.

The class II region of the human major histocompatibility complex (HLA) is made up of three major subregions designated DR, DQ, and DP. With the aim of gaining an insight into the evolution and stability of DR haplotypes, a total of 63 cosmid clones were isolated from the DR subregion (Gogo-DR) of a western lowland gorilla. All but one of these cosmid clones were found to fall into two clusters. The larger cluster, A, was defined by 41 overlapping cosmid clones and contained a DRB gene segment made up of exons 4 through 6 and four DRB genes, designated Gogo-DRB6, Gogo-DRB5*01, Gogo-DRB8, and Gogo-DRB3*01. The total length of this cluster was approximately 180 kb. The second cluster, B, encompassed a contiguous DNA stretch of approximately 145 kb and was composed of 21 overlapping cosmid clones. Cluster B contained three DRB genes, designated Gogo-DRB1*08, Gogo-DRB2, and Gogo-DRB3*02. One cosmid clone (WP1-9) containing a DRB pseudogene could not be linked to either cluster A or B. Neither the organization of cluster A nor that of cluster B was identical to that of known HLA-DR haplotypes. However, two gorilla DRB genes, Gogo-DRB6 and Gogo-DRB5*01, the human counterparts of which are linked in the HLA-DR2 haplotype, were found to be located next to each other in cluster A. The arrangement of the Gogo-DRB genes in cluster B, which is presumed to be the gorilla DR8 haplotype, was similar to that of HLA-DR3/DR5/DR6 haplotypes and to that of the presumed ancestral HLA-DR8 haplotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maize genome structure variation: interplay between retrotransposon polymorphisms and genic recombination

Although maize (Zea mays) retrotransposons are recombinationally inert, the highly polymorphic structure of maize haplotypes raises questions regarding the local effect of intergenic retrotransposons on recombination. To examine this effect, we compared recombination in the same genetic interval with and without a large retrotransposon cluster. We used three different bz1 locus haplotypes, McC, B73, and W22, in the same genetic background. We analyzed recombination between the bz1 and stc1 markers in heterozygotes that differ by the presence and absence of a 26-kb intergenic retrotransposon cluster. To facilitate the genetic screen, we used Ds and Ac markers that allowed us to identify recombinants by their seed pigmentation. We sequenced 239 recombination junctions and assigned them to a single nucleotide polymorphism-delimited interval in the region. The genetic distance between the markers was twofold smaller in the presence of the retrotransposon cluster. The reduction was seen in bz1 and stc1, but no recombination occurred in the highly polymorphic intergenic region of either heterozygote. Recombination within genes shuffled flanking retrotransposon clusters, creating new chimeric haplotypes and either contracting or expanding the physical distance between markers. Our findings imply that haplotype structure will profoundly affect the correlation between genetic and physical distance for the same interval in maize.     

Haplotype analysis of the apolipoprotein gene cluster on human chromosome 11

Members of the apolipoprotein gene cluster (APOA1/C3/A4/A5) on human chromosome 11q23 play an important role in lipid metabolism. Polymorphisms in both APOA5 and APOC3 are strongly associated with plasma triglyceride concentrations. The close genomic locations of these two genes as well as their functional similarity have hindered efforts to define whether each gene independently influences human triglyceride concentrations. In this study, we examined the linkage disequilibrium and haplotype structure of 49 SNPs in a 150-kb region spanning the gene cluster. We identified a total of five common APOA5 haplotypes with a frequency of greater than 8% in samples of northern European origin. The APOA5 haplotype block did not extend past the 7 SNPs in the gene and was separated from the other apolipoprotein gene in the cluster by a region of significantly increased recombination. Furthermore, one previously identified triglyceride risk haplotype of APOA5 (APOA5*3) showed no association with three APOC3 SNPs previously associated with triglyceride concentrations, in contrast to the other risk haplotype (APOA5*2), which was associated with all three minor APOC3 SNP alleles. These results highlight the complex genetic relationship between APOA5 and APOC3 and support the notion that APOA5 represents an independent risk gene affecting plasma triglyceride concentrations in humans.     

The identification and characterisation of alleles of sucrose phosphate synthase gene family III in sugarcane

Little is known about the extent of allelic diversity of genes in the complex polyploid, sugarcane. Using sucrose phosphate synthase (SPS) Gene (SPS) Family III as an example, we have amplified and sequenced a 400 nt region from this gene from two sugarcane lines that are parents of a mapping population. Ten single nucleotide polymorphisms (SNPs) were identified within the 400 nt region of which seven were present in both lines. In the elite commercial cultivar Q165^A, 10 sequence haplotypes were identified, with four haplotypes recovered at 9% or greater frequency. Based on SNP presence, two clusters of haplotypes were observed. In IJ76-514, a Saccharum officinarum accession, 8 haplotypes were identified with 4 haplotypes recovered at 13% or greater frequency. Again, two clusters of haplotypes were observed. The results suggest that there may be two SPS Gene Family III genes per genome in sugarcane, each with different numbers of different alleles. This suggestion is supported by sequencing results in an elite parental sorghum line, 403463-2-1, in which 4 haplotypes, corresponding to two broad types, were also identified. Primers were designed to the sugarcane SNPs and screened over bulked DNA from high and low Sucrose-containing progeny from a cross between Q165^A and IJ76-514. The SNP frequency did not vary in the two bulked DNA samples, suggesting that these SNPs from this SPS gene family are not associated with variation in sucrose content. Using an ecotilling approach, two of the SPS Gene Family III haplotypes were mapped to two different linkage groups in homology group 1 in Q165^A. Both haplotypes mapped near QTLs for increased sucrose content but were not themselves associated with any sugar-related trait.     

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.     

Determining the physical limits of the Brassica S locus by recombinational analysis

A genetic analysis was performed to study the frequency of recombination for intervals across the Brassica S locus region. No recombination was observed between the S locus glycoprotein gene and the S receptor kinase gene in the segregating populations that we analyzed. However, a number of recombination breakpoints in regions flanking these genes were identified, allowing the construction of an integrated genetic and physical map of the genomic region encompassing one S haplotype. We identified, based on the pollination phenotype of plants homozygous for recombinant S haplotypes, a 50-kb region that encompasses all specificity functions in the S haplotype that we analyzed. Mechanisms that might operate to preserve the tight linkage of self-incompatibility specificity genes within the S locus complex are discussed in light of the relatively uniform recombination frequencies that we observed across the S locus region and of the structural heteromorphisms that characterize different S haplotypes.     

Mapping of the major psoriasis-susceptibility locus (PSORS1) in a 70-Kb interval around the corneodesmosin gene (CDSN)

Numerous putative susceptibility loci have been described for psoriasis. Among the loci confirmed in the literature, PSORS1 (the major histocompatibility complex at 6p21.3) has the strongest effect. Recent studies have highlighted a 200-kb candidate region. However, this region has not been well delimited, mainly because of the strong linkage equilibrium among the associated alleles. To finely map PSORS1, we set up a study using 17 polymorphic markers in a 525-kb interval around the human leucocyte antigen C locus (HLA-C). The results uncovered five loci with alleles strongly associated with psoriasis (Sidak-corrected P [P(c)] values from 1.8 x 10(-7) to .003), all structured in a psoriasis-susceptibility haplotype (PSH). Subsequent analysis of extended haplotypes showed that the PSH was not only present on the traditional psoriasis-susceptibility extended haplotypes (HLA-Cw6-B57, HLA-Cw6-B37, and HLA-Cw6-B13) but also on a haplotype of Sardinian origin (HLA-Cw7-B58) found to be associated with psoriasis (Pc=.0009) because of an ancestral recombination with one of the susceptibility haplotypes carrying the HLA-Cw6 allele. Comparisons of the regions identical by descent among associated and nonassociated haplotypes highlighted a minimum region of 70 kb not recombinant with PSORS1, around the corneodesmosin (CDSN) gene.     

Extensive deletions in the Q region of the mouse major histocompatibility complex

By use of Southern blot analyses and low copy number probes, the fine structure of the Q region of the mouse major histocompatibility complex was studied in more detail. With a probe recognizing the even-numbered genes Q4, Q6, and Q8, it was evident that Q4 and/or the regions flanking Q4 are polymorphic, whereas Q6 and Q8, and their flanking regions are nonpolymorphic. Perhaps the most noteworthy finding is that at least two strain haplotypes, H-2 ^k and H-2 ^f, possessed extensive deletions in the Q region. The most striking deletion was found in the H-2 ^f haplotype, where the QI through Q9 genes appear to be missing. Because of these extensive deletions the functional importance of the Q region is questioned.     

Heterochromatic Stellate Gene Cluster in Drosophila Melanogaster: Structure and Molecular Evolution

The 30-kb cluster comprising close to 20 copies of tandemly repeated Stellate genes was localized in the distal heterochromatin of the X chromosome. Of 10 sequenced genes, nine contain undamaged open reading frames with extensive similarity to protein kinase CK2 β-subunit; one gene is interrupted by an insertion. The heterochromatic array of Stellate repeats is divided into three regions by a 4.5-kb DNA segment of unknown origin and a retrotransposon insertion: the A region (~14 Stellate genes), the adjacent B region (approximately three Stellate genes), and the C region (about four Stellate genes). The sequencing of Stellate copies located along the discontinuous cluster revealed a complex pattern of diversification. The lowest level of divergence was detected in nearby Stellate repeats. The marginal copies of the A region, truncated or interrupted by an insertion, escaped homogenization and demonstrated high levels of divergence. Comparison of copies in the B and C regions, which are separated by a retrotransposon insertion, revealed a high level of diversification. These observations suggest that homogenization takes place in the Stellate cluster, but that inserted sequences may impede this process.     

Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene.

The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiple of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1, 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between these alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations.     

Characteristics of the expression of the murine soluble class I molecule (Q10)

Q10 is a class I molecule previously proven to be secreted rather than membrane bound. To measure the amount of Q10 in various mouse sera, a quantitative Western blot assay was developed. Q10 was the only class I molecule detectable in mouse sera. It occurs as a high m.w. complex of 200,000 to 300,000. The amount of Q10 in serum varies among different mouse strains and is controlled by a region telomeric to H-2S. Mice of the f haplotype do not express Q10, but all other mice examined (20 strains) with inbred or wild-derived H-2 haplotypes do. The H-2 haplotypes rank according to their levels of Q10 as follows: z, s greater than k, b greater than d, q greater than f; and the actual values range from to 60 micrograms/ml to undetectable levels in serum. In some strains the levels are higher in males than in females. The levels increase with age and decrease during pregnancy but not during lactation. There is a dramatic decrease after the injection of irritants or syngeneic tumor transplantation, but allostimulation has no effect on Q10 levels. The possible significance of this soluble class I molecule is discussed in the light of our findings.     

Localization of psoriasis-susceptibility locus PSORS1 to a 60-kb interval telomeric to HLA-C

Recent genome scans have established the presence of a major psoriasis-susceptibility locus in the human leukocyte antigen (HLA) complex on chromosome 6p21.3. To narrow the interval for candidate gene testing, we performed a linkage-disequilibrium analysis of 339 families, with the use of 62 physically mapped microsatellite markers spanning the major histocompatibility complex (MHC). As detected by use of the transmission/disequilibrium test (TDT), individual markers yielded significant linkage disequilibrium across most of the MHC. However, the strongest evidence for marker-trait disequilibrium was found in an approximately 300-kb region extending from the MICA gene to the corneodesmosin gene. Maximum-likelihood haplotypes were constructed across the entire MHC in the original sample and across a 1.2-Mb region of the central MHC in an expanded sample containing 139 additional families. Short (two- to five-marker) haplotypes were subjected to the TDT using a "moving-window" strategy that reduced the variability of TDT P values relative to the single-locus results. Furthermore, the expanded sample yielded a sharp peak of evidence for linkage disequilibrium that spanned approximately 170 kb and that was centered 100 kb telomeric to HLA-C. The 1.2-Mb interval was further dissected by means of recombinant ancestral haplotype analysis. This analysis identified risk haplotype 1 (RH1), which is a 60-kb fragment of ancestral haplotype 57.1, on all identifiable HLA risk haplotypes. One of these haplotypes exhibits significant linkage disequilibrium with psoriasis but does not carry Cw6, which is the HLA allele most strongly associated with the disease. These results demonstrate that RH1 is highly likely to carry the disease allele at PSORS1, and they exclude HLA-C and corneodesmosin with a high degree of confidence.     

DNA polymorphism, haplotype structure and balancing selection in the Leavenworthia PgiC locus.

A study of DNA polymorphism and divergence was conducted for the cytosolic phosphoglucose isomerase (PGI:E.C.5.3.1.9) gene of five species of the mustard genus Leavenworthia: Leavenworthia stylosa, L. alabamica, L. crassa, L. uniflora, and L. torulosa. Sequences of an internal 2.3-kb PgiC gene region spanning exons 6-16 were obtained from 14 L. stylosa plants from two natural populations and from one to several plants for each of the other species. The level of nucleotide polymorphism in L. stylosa PgiC gene was quite high (pi = 0.051, theta = 0.052). Although recombination is estimated to be high in this locus, extensive haplotype structure was observed for the entire 2.3-kb region. The L. stylosa sequences fall into at least two groups, distinguished by the presence of several indels and nucleotide substitutions, and one of the three charge change nucleotide replacements within the region sequenced correlates with the haplotypes. The differences between the haplotypes are older than between the species, and the haplotypes are still segregating in at least two of five species studied. There is no evidence of recent or ancient population subdivision that could maintain distinct haplotypes. The age of the haplotypes and the results of Kelly's Z(nS) and Wall's B and Q tests with recombination suggest that the haplotypes are maintained due to balancing selection at or near this locus.     

tRNA2Lys gene clusters in Drosophila

We have isolated segments of Drosophila melanogaster DNA that contain two clusters of tRNA₂^Lys genes. In one segment, pPW511, there is a cluster of three of these genes surrounded by other tRNA genes. Two other segments, pPW516 and pPW541. share a 3 × 10³ base-pair region that has a cluster of four tRNA₂^Lys genes. This cluster is flanked by 20 × 10³ base-pairs of DNA that does not appear to have other tRNA genes. The tRNA genes in both clusters are irregularly spaced and are intermingled with moderately repetitive DNA. Each cluster is present once or perhaps twice in the haploid genome and has the same arrangement of restriction endonuclease sites in the genomic DNA as in the isolated, cloned DNA. In situ hybridization to polytene chromosomes localized the pPW511 cluster to the 42A region and the pPW516/541 cluster to the 42E region. Another region, 50B, also contains tRNA₂^Lys genes. In sum, these cloned tRNA₂^Lys genes account for most of this gene family and are irregularly spaced in two clusters.