Evidence for two B-cell alloantigen loci in theHLA-D Region

Antigenic determinants recognizable by human antisera (Hon 7 and 2075abs sera) were found in a partially purified antigen preparation obtained from an HLA-D and -DR homozygous cell line (EBV-Wa). Sequential coprecipition tests showed that two determinants detectable with Hon 7 and 2075abs sera (Hon 7 and 2075abs determinants) were present on different molecules. These two antigenic determinants were shown to be allotypic and were expressed predominantly in the B-cell-rich fraction. Family studies showed that both antigenic determinants segregated concordantly with respectiveHLA haplotypes. In the population study, the 2075abs and Hon 7 determinants were shown to be in strong linkage disequilibrium and the 2075abs determinant perfectly correlated with the HLA-DRw4 specificity. The results indicate that the Hon 7 determinant is coded for by a gene distinct from alleles at theHLA-DR locus. Furthermore, the locus (Hon 7) coding for the Hon 7 determinant is suggested to be very closely linked with theHLA-DR locus.     

Use of immunochemical and SSCP analyses to test homozygosity at theS locus ofBrassica oleracea genotypes

The self-incompatibility reaction of cruciferous plants prevents self-fertilization and has been shown to be controlled by at least two genes situated at a single multiallelic locus, theS locus. One of these two genes, theS locus glycoprotein (SLG) gene, encodes an abundant glycoprotein secreted to the cell wall of stigma papillae. Identification of thoseS alleles present at theS locus is of prime interest when studying the self-incompatibility response and can be achieved by identifying the SLG of the stigma. Here, we show that using anti-SLG antibodies in an immunochemical analysis, combined with a SSCP (single-strand conformation polymorphism) approach to characterize the corresponding stigma-specific, SLG mRNA, allowed the identification of plants heterogeneous at theS locus among populations of plants that were thought to be homozygous for known SLG alleles. This analysis stresses the importance of testing the homozygosity at theS locus of lines considered inbred for a knownS allele as mix-up of seeds may occur during the breeding programme.     

TheD17Tu5 locus in thet complex: implications for the origin oft haplotypes and inbred strains

The mouse × Chinese hamster cell line R4 4-1 contains only one mouse chromosome, the bulk of which corresponds toMus musculus chromosomes 17 and 18 (MMU17 and MMU18, respectively). A genomic library was prepared from the R4 4-1 DNA, and a mouse clone was isolated from the library, which—with the help of somatic cell hybrids-could be mapped to the MMU17. A locus defined by a 2.7-kb longBam HI probe from this clone was designatedD17Tu5 (Tu for Tübingen). The locus proved to be polymorphic among inbred strains and wild mice. By testing of recombinant inbred strains and partialt haplotypes, theD17Tu5 locus could be mapped to a position between theD17Leh66E andD17Rp17 loci within thet complex. Two alleles were found at this locus,D17Tu5 ^a andD17Tu5 ^b , defined byTaq I restriction fragment length polymorphism. Both alleles are present among inbred strains and wild mice of the speciesM. domesticus. All completet haplotypes tested carry theD17Tu5 ^a allele and all tested wild mice of the speciesM. musculus, with the exception of those bearingt haplotypes, carry theD17Tu5 ^b allele. Additional alleles are found in some populations of wild mice and in other species of the genusMus. The distribution of the two alleles among the inbred strains correlates well with their known or postulated genealogy. Their distribution between the two species ofMus and among the mice withT haplotypes suggests a relatively recent origin of thet haplotypes.     

Evolutionary stability of MHC class II haplotypes in diverse rhesus macaque populations

A thoroughly characterized breeding colony of 172 pedigreed rhesus macaques was used to analyze exon 2 of the polymorphic Mamu-DPB1, -DQA1, -DQB1, and -DRB loci. Most of the monkeys or their ancestors originated in India, though the panel also included animals from Burma and China, as well as some of unknown origin and mixed breeds. In these animals, mtDNA appears to correlate with the aforementioned geographic origin, and a large number of Mamu class II alleles were observed. The different Mamu-DPB1 alleles were largely shared between monkeys of different origin, whereas in humans particular alleles appear to be unique for ethnic populations. In contrast to Mamu-DPB1, the highly polymorphic -DQA1/DQB1 alleles form tightly linked pairs that appear to be about two-thirds population specific. For most of the DQA1/DQB1 pairs, Mamu-DRB region configurations present on the same chromosome have been ascertained, resulting in 41 different -DQ/DRB haplotypes. These distinct DQ/DRB haplotypes seem to be specific for monkeys of a determined origin. Thus, in evolutionary terms, the Mamu-DP, -DQ, and -DR regions show increasing instability with regard to allelic polymorphism, such as for -DP/DQ, or gene content and allelic polymorphism, such as for -DR, resulting in population-specific class II haplotypes. Furthermore, novel haplotypes are generated by recombination-like events. The results imply that mtDNA analysis in combination with Mhc typing is a helpful tool for selecting animals for biomedical experiments.The sequences reported in this paper have been deposited in the EMBL database (accession nos. AJ534296–AJ534304, AJ 564564, and AJ557455–AJ557511)     

A polymorphic system related to but genetically independent of the chicken major histocompatibility complex

Analyses of the major histocompatibility complex (Mhc) in chickens have shown inconsistencies between serologically defined haplotypes and haplotypes defined by the restriction fragment patterns of Mhc class I and class II genes in Southern hybridizations. Often more than one pattern of restriction fragments for Mhc class I and/or class II genes has been found among DNA samples collected from birds homozygous for a single serologically defined B haplotype. Such findings have been interpreted as evidence for variability within the Mhc haplotypes of chickens not detected previously with serological methods. In this study of a fully pedigreed family over three generations, the heterogeneity observed in restriction fragment patterns was found to be the result of the presence of a second, independently segregating polymorphic Mhc-like locus, designated Rfp-Y. Three alleles (haplotypes) are identified in this new system.     

The “Sardinian”HLA-A30,B18,DR3,DQw2 haplotype constantly lacks the21-OHA andC4B genes. Is it an ancestral haplotype without duplication?

TheC4 and21-OH loci of the class III HLA have been studied by specific DNA probes and the restriction enzymeTaq I in 24 unrelated Sardinian individuals selected from completely HLA-typed families. All 24 individuals had theHLA extended haplotypeA30,Cw5,B18, BfF1,DR3,DRw52,DQw2, named “Sardinian” in the present paper because of its frquency of 15% in the Sardinian population. Eighteen of these were homozygous for the entire haplotype, and six were heterozygous at theA locus and blank (or homozygous) at all the other loci. In all completely homozygous cells and in four heterozygous cells at theA locus, the restriction fragments of the21-OHA (3.2 kb) andC4B (5.8 kb or 5.4 kb) genes were absent, and the fragments of theC4A (7.0 kb) and21-OHB (3.7 kb) genes were present. It is suggested that the “Sardinian” haplotype is an ancestral haplotype without duplication of theC4 and21-OH genes, practically always identical in its structure, also in unrelated individuals. The diversity of this haplotype in the class III region (about 30 kb less) may be at least partially responsible for its misalignment with most haplotypes, which have duplicatedC4 and21-OH genes, and therefore also for its decreased probability to recombine. This can help explain its high stability and frequency in the Sardinian population. The same conclusion can be suggested for the Caucasian extended haplotypeA1,B8,DR3 that always seems to lack theC4A and21-OHA genes.     

A premature stop codon in the TYRP1 gene is associated with brown coat colour in the European rabbit (Oryctolagus cuniculus)

Classical genetic studies in European rabbits (Oryctolagus cuniculus) suggested the presence of two alleles at the brown coat colour locus: a wild‐type B allele that gives dense black pigment throughout the coat and a recessive b allele that in the homozygous condition (b/b genotype) produces brown rabbits that are unable to develop black pigmentation. In several other species, this locus is determined by mutations in the tyrosinase‐related protein 1 (TYRP1) gene, encoding a melanocyte enzyme needed for the production of dark eumelanin. In this study, we investigated the rabbit TYRP1 gene as a strong candidate for the rabbit brown coat colour locus. A total of 3846 bp of the TYRP1 gene were sequenced in eight rabbits of different breeds and identified 23 single nucleotide polymorphisms (SNPs; 12 in intronic regions, five in exons and six in the 3′‐untranslated region) and an insertion/deletion of 13 bp, in the 3′‐untranslated region, organised in a few haplotypes. A mutation in exon 2 (g.41360196G>A) leads to a premature stop codon at position 190 of the deduced amino acid sequence (p.Trp190ter). Therefore, translation predicts a truncated TYRP1 protein lacking almost completely the tyrosinase domain. Genotyping 203 rabbits of 32 different breeds identified this mutation only in brown Havana rabbits. Its potential functional relevance in disrupting the TYRP1 protein and its presence only in brown animals strongly argue for this non‐sense mutation being a causative mutation for the recessive b allele at the brown locus in Oryctolagus cuniculus.     

Segregation of allotypes in a strain of chickens homozygous for theB locus

Sublines homozygous for alleles controlling IgM and IgG allotypes at two linked loci (M1 andG1) were established in the B14 strain of chickens, which is homozygous for theB locus controlling major histocompatibility antigens. Transfer of lymphoid cells resulted in a permanent donor-type IgG synthesis in irradiated 12-day-old recipients, but was followed by protracted rejection in chickens injected embryonally.     

Genetic control of lymphocyte antigens in sheep: TheOLA complex and two minor loci

Lymphocytotoxic reagents allowed us to define 11 lymphocyte factors in sheep. After a genetic control of monospecificity had been made, a genetic study based on 400 crossings indicated that nine factors are transmitted in two haplotypes containing 0.1 or 2 factors. The linked factors of the bifactorial haplotypes are the products of two genes at two closely linked loci,OLA- A andOLA- B, having a recombination rate of about 0.6%. The nine factors are the products of fiveOLA- A and fourOLA- B alleles. Allelic frequencies at the two loci, ranging from 0.05 to 0.23 were obtained by two methods. A linkage disequilibrium between the loci is described. Among the possible causes of this, inbreeding is excluded, whereas selective pressure or more probably old fusions between sheep populations must be retained. A tenth factor (frequency 0.33 to 0.35) has a 26% recombination with OLA factors; its locus (OL-X) would be on the same chromosome but very far from theOLA complex. An 11th factor (frequency 0.30) is probably independent ofOLA; its locus (OL-Z) may be on another chromosome. The two linkedOLA loci give evidence of a major histocompatibility complex in sheep; the observed linkage disequilibrium allows one to foretell certain applications in phylogeny and perhaps in selection.     

The effects of heterozygosity at the af,st and tl loci in peas

Summary Eight near-isogenic lines of pea representing all the homozygous combinations of three genes af, st and tl, which modify leaf shape and size, were crossed in all possible ways excepting reciprocals. An analysis of the resulting 36 families has shown that homozygous mutant alleles at the tl locus acting with homozygous mutant alleles at the af and st loci increase both seed weight and plant haulm weight. The mutant alleles at the af and st loci seem, when homozygous, to have little effect by themselves upon seed weight but they do increase or decrease haulm weight, respectively. There is clear evidence of heterotic effects resulting from heterozygosity at each one of the three loci which modify seed weight, haulm weight and basal branching. The implications of such heterotic effects in pea breeding programmes are discussed.     

S-related protein can be recombined with self-compatibility in interspecific derivatives ofLycopersicon

Stylar proteins involved in the self-incompatible (SI) response ofLycopersicon hirsutum have been identified and mapped to the locus that controls SI (S locus).L. esculentum, a self-compatible (SC) species of cultivated tomato, does not display these proteins. Hybrids between SCL. esculentum and SIL. hirsutum are self-sterile despite these individuals bearing pollen containing theS allele ofL. esculentum. In progeny derived from backcrossing the hybrids toL. esculentum, there was a strong correlation between the presence of theS allele fromL. hirsutum and self-infertility. However, this relationship was uncoupled in a number of backcross (BC) progeny. The SI response appeared to be nonexistent in two self-fertile BC individuals that were heterozygous for theS allele ofL. hirsutum, based on Mendelian segregation of a tightly linked DNA marker,CD15, in selfed progeny. Among these progeny self-fertile individuals that were homozygous for theL. hirsutum allele of the linked marker were also determined to be homozygous for anS-related protein ofL. hirsutum through test crosses withL. esculentum. Therefore, plants were produced that were homozygous for a functionalS allele but were self-fertile. This result and other evidence suggest that theS-related proteins are not sufficient to elicit a self-incompatible response inL. esculentum and that there is a mutation(s) inL. esculentum somewhere other than theS locus that leads to self-compatibility.     

Homoeosis in Drosophila: Evidence for a maternal effect of the polycomb locus

When heterozygous, dominant mutant alleles of the Polycomb locus are associated with a variety of adult homoeotic effects. Zygotes homozygous for these alleles die as late embryos showing homoeotic transformation of head, thoracic, and abdominal segments. This study shows that embryos homozygous for Pc³ are more extreme than those homozygous for Pc¹ or Pc². Moreover, Pc¹/Pc³ heterozygotes are more extensively transformed if their mothers were Pc³/ + than if they were Pc¹/ +; this effect does not depend on zygotic genetic background and must be maternal in nature. Embryos homozygous for Pc³ are less extreme if they arise from Pc³/ + / + than from Pc³/ + mothers. These results strongly suggest that the Polycomb locus acts maternally as well as zygotically to affect early determinative decisions.     

Haplotypic variation of the transporter associated with antigen processing (TAP) genes and their extension of HLA class II region haplotypes

Stable cell surface presentation of HLA class I molecules requires active transport of antigenic peptides across the endoplasmic reticulum by products of two genes, TAP1 and TAP2, which map in the major histocompatibility complex class II region. Alleles of each gene are derived from a combination of variable sitesaat each locus. In this study, TAP1 and TAP2 alleles were identified in homozygous typing cell (HTC) lines, allowing resolution of specific haplotypes in conjunction with the highly polymorphic HLA class II region haplotypes. Three alleles at each TAP locus were found from which eight haplotypes could be assigned. Determination of TAP1 and TAP2 alleles in cell lines homozygous at DR, DQ, and DP created eight additional haplotypes beyond the number observed with these class II genes alone. Complete analysis of DR, DQ, TAP, and DP genotypes in 66 HTCs resulted in the following groups: 1) 46 homozygotes; 2) nine homozygous at DR, DQ, and TAP, but heterozygous at DP; 3) four homozygous at DR, DQ, and DP, but heterozygous at one or both TAP genes; 4) four homozygous at DR and DQ, but heterozygous at TAP and DP; and 5) three complex genotypes heterozygous at DP, TAP, and at least one of DQA1, DQB1, or DRB1 loci. TAP1 and TAP2 genes map in an area of frequent recombination. TAP alleles were determined in five DQB1, DPB1 recombinant individuals, three of which were informative. Recombination was found between DQB1 and the TAP loci in two individuals and between TAP and DPB1 in the other individual.     

The biallelica mating type locus ofUstilago maydis: remnants of an additional pheromone gene indicate evolution from a multiallelic ancestor

Thea mating type locus ofUstilago maydis contains the structural genes for a pheromone-based cell recognition system that governs fusion of haploid cells. The locus exists in two alleles, termeda1 anda2. We have completed the analysis of the nucleotide sequences unique toa1 anda2. Within these dissimilar regions we find two short patches of DNA sequence similarity. Interestingly, one of these segments corresponds to the transcribed region of thea1 pheromone precursor. As a result of multiple nucleotide exchanges this sequence does not code for a functional product. The existence of a second pheromone gene in thea2 allele suggests that the present locus had a multiallelic ancestor. In addition, we describe the presence of two additional genes in thea2 allele. We have investigated the role of these genes during mating and pathogenic development and speculate that they might affect mitochondrial inheritance.     

Monophyletic origin of naked barley inferred from molecular analyses of a marker closely linked to the naked caryopsis gene (<Emphasis Type="Italic">nud</Emphasis>)

To elucidate the origin of naked barley, molecular variation of the marker sKT7 tightly linked to the nud locus was examined. A total of 259 (53 wild, 106 hulled domesticated, and 100 naked domesticated) barley accessions were studied. Restriction analysis of the sKT7 PCR-amplified product revealed the alleles I, II, III, and IV. All four alleles were found in wild barley, but allele IV was found only in a single accession from southwestern Iran. Hulled domesticated accessions showed alleles I, II, or III, but all naked domesticated accessions had allele IV. The distribution of allele IV in wild barley and its pervasive presence in naked domesticated lines support the conclusion that naked barley has a monophyletic origin, probably in southwestern Iran. The available results suggest two scenarios for the origin of naked barley: either directly from a wild barley with allele IV or from a hulled domesticated line with allele IV that later became extinct. Naked domesticated accessions from different regions of the world have extremely homogeneous DNA sequences at the sKT7 locus, supporting the monophyletic origin of naked barley. For allele IV, four haplotypes (IVb to IVe) were found in 30 naked accessions: IVb was predominant (66.7%) and widely distributed, while the other three haplotypes, differing by only one nucleotide at different positions relative to IVb, showed a localized distribution. The geographical distribution of the haplotypes of sKT7 allele IV suggests migration routes of naked domesticated barley in central and eastern Asia.Communicated by F. Salamini     

Angiotensin I-converting enzyme (ACE): estimation of DNA haplotypes in unrelated individuals using denaturing gradient gel blots

The angiotensin I-converting enzyme (ACE) gene (17q23) is a candidate gene for essential hypertension and related diseases, but investigation of its role in human pathology is hampered by a lack of identified polymorphisms. Currently, a 287-bp insertion/deletion (I/D) RFLP in intron 16 represents the only one known. Additional polymorphisms for the ACE gene would make most families informative for linkage studies and would allow haplotypes to be assigned in association studies. To increase the information provided by the ACE gene, we used a sensitive screening technique, denaturing gradient gel electrophoresis (DGGE) blots, to identify polymorphisms and combined this with gene counting to identify haplotypes. Five independent polymorphisms, restriction fragment melting polymorphisms (RFMPs), were identified by four probes (encompassing half of the ACE cDNA) in digests produced by three restriction enzymes (DdeI, RsaI, and AluI). One RFMP has three alleles while the others have two alleles. In a sample of 67 unrelated control subjects, minor allele frequencies ranged from 0.12 to 0.49. A significant level of linkage disequilibrium was found for all pairs of markers. The four most informative RFMPs, taken in combination, define 24 potential haplotypes. Based on gene counting, 11 of the 24 are rare or nonexistent in this population, and the estimated heterozygosity of the remaining 13 haplotypes approaches 80%. Under these conditions for the ACE locus, phase-unknown genotypes could be assigned to haplotype pairs in unrelated subjects with reasonable certainty. Thus, using DGGE blot technique for identifying numerous DNA polymorphisms in a candidate locus, in combination with gene counting, one can often identify DNA haplotypes for both related and unrelated study subjects at a candidate locus. These markers in the ACE gene should be useful for clinical and epidemiologic studies of the role of ACE in human disease.     

FB elements can promote exon shuffling: a promoter-less white allele can be reactivated by FB mediated transposition in Drosophila melanogaster

Foldback (FB) elements are transposable elements found in many eukaryotic genomes; they are thought to contribute significantly to genome plasticity. In Drosophila melanogaster, FBs have been shown to be involved in the transposition of large chromosomal regions and in the genetic instability of some alleles of the white gene. In this report we show that FB mediated transposition of w ^67C23, a mutation that deletes the promoter of the white gene and its first exon, containing the start codon, can restore expression of the white gene. We have characterized three independent events in which a 14-kb fragment from the w ^67C23 locus was transposed into an intron region in three different genes. In each case a local promoter drives the expression of white, producing a chimeric mRNA. These findings suggest that, on an evolutionary timescale, FB elements may contribute to the creation of new genes via exon shuffling.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. P. Georgiev     

The HLA-D determinants

Stimulating cells assumed to be homozygous at theHLA-D locus were used to identify nine different MLC-activating HLA-D determinants in a population of unrelated Norwegians. The results suggest that the HLA-D determinants typed for, which account for a combined gene frequency of approximately 0.80, are controlled by mutually exclusive codominant alleles at one locus. Some of the HLA-D determinants showed highly significant association to certain HLA-B antigens.     

Stable expression of a single-copy rolA gene in transgenic Arabidopsis thaliana plants allows an exhaustive mutagenic analysis of the transgene-associated phenotype

Several publications have documented the instability of transgene expression in plants. Previous genetic approaches to the study of transgene-associated phenotypes in plants were limited by this phenomenon. Here we show that a transgene can be expressed in plants with sufficient stability to allow an exhaustive mutagenic analysis of the resulting phenotype. We have expressed the morphogenic rolA gene from the TL-DNA of Agrobacterium rhizogenes Ri plasmid in transgenic Arabidopsis thaliana plants. The resulting pleiotropic RolA phenotype allows a visual screen for reversion to detect germinal as well as somatic instability of transgene expression. However no spontaneous reversions of the RolA phenotype were observed in 65 000 progeny of two independent transgenic A. thaliana lines, each carrying a single homozygous rolA locus. In contrast, 12 revertants of the RolA phenotype were isolated from 360000 ethyl methane sulphonate (EMS)-mutagenized M2 progeny. All revertants were shown genetically to carry stable recessive mutations in the rolA locus, thus establishing a series of loss-of-function alleles. Molecular characterization revealed that the loss-of-function alleles were structurally intact and expressed in all rolA mutants. A wild-type rolA locus and two loss-of-function alleles were reisolated and sequenced; base pair substitutions were found in each loss-of-function allele leading to single amino acid substitutions in the rolA open reading frame. Therefore no instability of expression of the rolA locus was detected in any of the 425 000 individuals studied in this analysis. Furthermore even under conditions of saturation mutagenesis, no extragenic suppressor locus was detected.     

Polymorphic population genetic markers for the Australian wood cockroach Panesthia australis

Primers for five polymorphic nuclear DNA (nDNA) markers and one mitochondrial DNA (mtDNA) gene (COI) were developed for the Australian wood cockroach Panesthia australis using a range of techniques. Eight mitochondrial haplotypes and four to 36 alleles per nuclear locus were detected in 744 cockroaches. Observed heterozygosity ranged from 0.117 to 0.816 in a sample of 30 animals from one population. The markers will be useful for population biology and for the measurement of the effects of habitat fragmentation on this ecologically important forest‐dependent species.