A cellular and functional split in the DRw8 haplotype is due to a single amino acid replacement (DR β ser 57- asp 57 )

The single DR beta chain gene of the DRw8 haplotype has been suggested to carry both the DRw8 and the DRw52 epitopes. Cellular typing has shown that the DRw8 haplotype can be split into three subtypes, Dw8.1. Dw8.2, and Dw8.3, presumably due to a polymorphism in the DRw8 chain. Furthermore, Dw8.1 and Dw8.2 cells present influenza virus antigen to different T-cell clones. In the present study, DRw8/Dw8.2 chain cDNA was cloned and characterized. A comparison of this sequence with a partial DRw8/Dw8.1 chain gene suggested that the DRw8 split is due to a single amino acid replacement of ser ₅₇ -asp ₅₇ caused by three nucleotide substitutions in the same codon. In most DR haplotypes, two expressed DR beta chain genes exist. Comparing the nucleotide sequence of the single beta gene in the DRw8 haplotype to those of other DR beta genes revealed that the DRw8 beta gene sequence is most closely related to the DRBI genes of the DR3, 5, and w6 haplotypes. However, the comparisons also showed that it was not possible from sequence similarities to divide the DR beta genes into two or more distinct allelic series.     

Sequence analysis and HLA-DR genotyping of a novel HLA-DRw14 allele

Analysis of a Japanese population by oligonucleotide genotyping revealed that one Japanese HLA-DRw14 allele had a DRB1 genotype different from that of the known HLA-DRw14-related alleles, DRB1 ^* 1401 (DRw14-Dw9) and DRB1 ^* 1402 (DRw14-Dw16). The second exon of the DRB1 gene of the novel DRw14 allele (designated DRB1-14c) was amplified enzymatically and sequenced after cloning intto a plasmid vector. The amino acid sequence of the first domain in the DR1 chain encoded in the DRB1-14c allele was more similar to that of the DRB1 ^* 1401 allele (three amino acid substitutions).than to that of the DRB1 ^* 1402 allele (six amino acid substitutions). No polymorphic amino acid residue that could explain the common serologic HLA-DRw14 specificity was identified among the sequences of the three DRw14-related alleles. Sequence-specific oligonucleotides (SSOs) were synthesized on the basis of the DRB1-14c nucleotide sequence and used for genotyping of the Japanese population. These SSOs served as useful probes for identifying the DRB1-14c allele in a wide range of donors.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M33693.     

Oligonucleotide genotyping shows that alleles at the HLA-DR β III locus of the DRw52 supertypic group segregate independently of known DR or Dw specificities

Using locus- and allele-specific oligonucleotide probes, we have studied the polymorphism of the HLA-DR III locus within the haplotypes of the DRw52 supertypic group. DNA from a number of homozygous typing cells typed for both Dw and DR was used. The DR III polymorphisms, DRw52a and DRw52b, do not segregate with Dw typing, or with DR typing, indicating that the determinants responsible for Dw-defined T -cell response and for DR haplotypic recognition are not encoded by the DR III locus. Hence, we can conclude that these DR specificities are encoded by the other functional DR locus, DR I, while the DR III locus encodes only the supertypic product.     

The narcolepsy-associatedDRw15, DQw6, Dw2 haplotype has no uniqueHLA-DQA or -DQB restriction fragments and does not extend to theHLA-DP subregion

Almost all patients with cataplectic narcolepsy are DR2-positive. It has been suggested that thenon-DR2 allele/haplotype might not be neutral with respect to disease susceptibility. It has also been reported thatTaq IDQA andBam HI,Eco RI,Eco RV, andPst IDQB restriction fragments might differentiate between narcoleptic and healthy DR2-positive individuals. In the present study,HLA class II gene polymorphisms were investigated by restriction fragment length polymorphism (RFLP) analysis in 47 Swedish patients with cataplectic narcolepsy, 100 random controls, and DR2-associated homozygous cell lines. All patients hadTaq IDRBDQA-DQB patterns corresponding to theDRw15,DQw6, Dw2 haplotype. The non-DR2 haplotype was found to be neutral. This genotyped group of patients allows firm rejection of a recessive mode of inheritance and supports a dominant or additive model. NoDQA orDQB RFLPs were found that could differentiate between DR2-positive narcoleptics, DRw15,DQw6,Dw2-positive controls, orDw2-homozygous cell lines. No significantMsp IHLA-DP association was found. No linkage disequilibrium was observed between theDRw15,DQw6,Dw2 haplotype and alleles of theDP subregion in patients or controls. Thus, theHLA-D region-associated narcolepsy susceptibility gene may be located telomeric to theHLA-DP subregion. No RFLPs have been observed that can locate the narcolepsy susceptibility gene closer to theDQ than to theDR subregion.     

DNA typing of anHLA-DR bilocus specificity by gene amplification and oligonucleotide hybridization

The structure of theDRB1 ^* 03 gene has been interpreted as the product of a gene conversion event involving aDRB3 gene as donor and resulting in the introduction of two short segments of the DRB3 sequence into theDRB1 locus. The serological counterpart of this double insertion is the TR81 specificity. Consequently, the TR81-specifying sequences can reside on eitherDRB1 orDRB3, or on both loci. Within each of the two sequence stretches a single nucleotide may be responsible for the generation of the TR81 alloantigen. Oligonucleotide probes corresponding to these stretches and to their allelic variants were constructed. They were used, under stringent hybridization conditions, to detect TR81-specifying sequences in the DNA ofHLA-homozygous cell lines carrying different haplotypes of the DRw52 family. Prior to hybridization the DNA was amplified with either DRB1-specific or DRB3-specific primers. Using this approach it was possible to perform a DNA typing of the TR81-specifying sites separately on both theDRB1 locus and theDRB3 locus.     

Ten HLA-DR4 alleles defined by sequence polymorphisms within the DRB1 first domain

We have studied DRB1 sequence polymorphisms associated with DR4 subtypes using DR4-specific DNA amplification and sequence-specific oligonucleotide probe (SSOP) hybridization. The 5 amplification primer was designed to hybridize with a unique sequence in the first hypervariable region (HVR) of the DRB1 second ex-on of all known DR4 alleles; the 3 primer was designed to hybridize with an intron sequence common to all DRB1 alleles. The specificity of the amplification step was demonstrated by double-blind testing of 105 selected DNA samples. Prospective SSOP typing of DR4 alleles was performed in 104 unrelated individuals known to be DR4-positive, including 13 who were DR4-homozygous. A DRB1 subtype corresponding with the previously defined DR4-associated specificities Dw4, Dw10, Dw13.1, Dw13.2, Dw14.1, Dw14.2, Dw15, and DwKT2 could be assigned for each of the 117 DR4 haplotypes tested. In most cases, DR4-homozygous, DRB1-heterozygous individuals could be genotyped with the panel of probes. In the course of our analysis, we identified two new DR4-related alleles, DRB1*04.CB (DRB1*0410)¹ and DRB1*04.EC (DRB1*, 0411)² which were recognized by their novel hybridization patterns. The DRB1 second exon sequence of DRB1*04.CB, is identical to DRB1*0405 except at codon 86 where GTG encodes valine instead of GGT encoding glycine. DRB1*04.EC is identical to DRB1*04.CB except at codon 74 where GAG encodes glutamic acid instead of GCG encoding alanine. Our results provide further evidence that SSOP hybridization is the most effective approach available for subtyping DR4 haplotypes and identifying unrecognized variants. A similar approach should be equally informative for subtyping other DR alleles.     

Polymorphisms within the HLA-DRw6 haplotype

We have analyzed the HLA class II gene products from HLA-DRw6 homozygous cells. Epstein-Barr virus-transformed B-cell lines were internally labeled with [³⁵S]-methionine. An NP-40 lysate of the cells was subjected to immunoprecipitation, first with a DRw52-like-specific monoclonal antibody and subsequently with a DR-specific framework antibody. The DR region-encoded gene products were analyzed by one-dimensional gel isoelectric focusing and two-dimensional gel electrophoresis. It is shown that DRw6 homozygous cell lines contain at least two nonallelic DR chains, one carrying a DRw52 determinant and one DRw52-negative population. Both chains appear to be polymorphic between the cellularly defined subtypes of DRw6. The determinant responsible for the differential mixed lymphocyte culture reactivity of Dw18 and Dw19 cells resides on the DRw52-positive population, whereas the Dw6-Dw9 differences are attributed to determinants on both populations of DR light chains. The Dw16-derived DRw52⁺ chain much resembles the Dw18 DRw52⁺ light chain whereas there is a clear-cut difference between these two subtypes in the DRw52^– population. We conclude that, for DRw6 homozygous cells, the cellularly recognized D determinants are probably located on DR-encoded molecules, both DRw52⁺ and DRw52^–, and that charge shift of these chains is at least partly responsible for differential recognition of these cells in mixed lymphocyte cultures.Abbreviations used in this paper: MLC mixed lymphocyte culture - 1D-IEF one-dimensional isoelectric focusing - 2D two-dimensional - moab monoclonal antibody     

Structural comparison of the genes of two HLA-DR supertypic groups: The loci encoding DRw52 and DRw53 are not truly allelic

The organization and sequence of the HLA-DR chain genes are compared in the two supertypic groups, DRw52 and DRw53, which together account for more than 80% of HLA-DR alleles. From the structural data, we conclude that these two groups represent distinct lineages which have followed different patterns of evolution. The fine structure of the chain locus encoding the DRw53 specificity corresponds most closely to the DR II pseudogene in the DRw52 haplotypes. Concomitantly, the DR I locus in DRw53 haplotypes is more closely related to both of the two expressed DR loci of theDRw5 haplotypes (DR I and DR III). These two loci are the result of a recent duplication. This leads to the proposal that both expressed DR chain genes in the DRw52 haplotypes (DR I and DR III) are derived from a single precursor locus, while the two loci expressed in the DRw53 haplotypes are derived from distinct ancestral loci. The genes encoding DRw52 and DRw53 are therefore not true alleles of the same original locus. A scheme is proposed that accounts for the evolution of DR specificities within the DRw52 and DRw53 groups of haplotypes. It is evident that the differentHLA-DR alleles are not structurally equidistant and that one must take into consideration different degrees of heterozygosity or mismatch among the DR alleles.     

Characterization of an HLA-DR β pseudogene in the DRw52 supertypic group

The nature of the DR II pseudogene in a haplo-type of the DRw52 supertypic group was investigated by nucleotide sequence analysis. It revealed several deleterious mutations in the signal sequence and second domain regions in addition to the complete absence of the first domain and adjacent sequences. No expression of DR II pseudogene mRNA can be detected. The same DR II pseudogene is probably present in other members of the DRw52 supertypic group. The pattern of mutations in this DR II pseudogene is different from that observed in the DR pseudogene of the DRw53 supertypic group, indicating a distinct evolutionary pathway for these two groups of DR haplotypes.     

Two divergent routes of evolution gave rise to the DRw13 haplotypes

The HLA class II genes and haplotypes have evolved over a long period of evolutionary time by mechanisms such as gene conversion, reciprocal recombination and point mutation. The extent of the diversity generated is most clearly evident in an analysis of the HLA class II alleles present within DRw13 haplotypes. This study uses cDNA sequencing to examine the first domains of DRB1, DRB3, DQA1, and DQB1 alleles from several American black individuals expressing seven different DRw13 haplotypes, five with undefined HLA-D specificities (i.e., not Dw18 or Dw19). Two new DRw13 alleles described in this study are the first examples of convergent evolution of DR alleles in which gene conversion has apparently combined segments of DRB1 alleles encoding DRw11 and DRw8 to generate two new DRB1 alleles, DRB1*1303 and DRB1*1304, that encode molecules bearing serologic determinants of a third allele, DRw13. These new DRw13 alleles are found embedded in haplotypes of DRw11 origin distinct from haplotypes encoding previously identified DRw13 alleles, DRB1*1301 and DRB1*1302. These data suggest that two evolutionary pathways may have given rise to two subgroups of alleles encoding molecules that share DRw13 serologic determinants yet which possess different structural and, likely, functional motifs. Reciprocal gene recombination events resulting in different DR, DRw52 and DQ allele combinations also appear to have played a crucial role in augmenting the level of diversity found in DRw13 haplotypes. Recombination has resulted in the association of one of the new DRw13 alleles with a DQw2 allele normally found associated with DR7 and the association of the DRw52c-associated DRw13 allele (DRB1*1302) with three different DQw1 alleles. The seven DRw13 haplotypes that have resulted from the effect of recombination on haplotypes formed by the two pathways of DRw13 allelic diversification have resulted in different repertoires of class II molecules and, most likely, different immune response profiles in individuals with these haplotypes.     

Heterogeneity inHLA-DR2-relatedDR,DQ haplotypes in eight populations of Asia-Oceania

The relative distributions of 480DR2-relatedDR, DQ haplotypes have been determined in Australian Aborigines, Papua New Guinean Highlanders, coastal Melanesians, Micronesians, Polynesians, Javanese, and Southern and Northern Chinese. Using sequence-specific oligonucleotides (SSOs) for hybridization of polymerase chain reaction (PCR) products from DRBI,DRBS,DQA1, andDQBI genes, 15 differentDR2-related haplotypes were identified. The predominantDR2 haplotype in Oceania involved a novel combination ofDRBI ^* 1502,DRB5 ^* 0101 alleles; this haplotype occurred sporadically in Java, but not in China. In Southern China, the most frequent DR2 haplotype involved the unusual arrangementDRB1 ^* 1602,DRB5 ^* 0101; alternatively,DRB1 ^* 1602 was associated with a newDRB5 SSO pattern. This study has important implications for molecular HLA-typing protocols that assume particularDRB1 DRB5 orDR,DQ linkage relationships. Further, the novelDRBI,DRB5 haplotype in Oceania suggests that the mixed lymphocyte culture (MLC) determinants Dw2 and Dw12 are discriminated by codon 86 at theDRBI locus.     

Mapping and nucleotide sequence of a newHLA class II light chain gene,DQB3

A genomic clone specifying a new HLA class II antigen β chain,DQB3, was isolated from a human genomic phage library using aDQB1 cDNA probe under low stringency conditions. Southern hybridization and nucleotide sequence analyses identified the β2 domain exon (exon 3) with several deleterious mutations and the CP-TM-CY exon [connecting peptide, transmembrane, and cytoplasmic regions, (exon 4)], but the first, second, and fifth exons encoding the 5′ UT-leader, the β1 domain, and the 3′ UT domain of normal β chains, respectively, were entirely missing. The nucleotide sequences of these two exons were distinct from those of other class II β chain genes, but slightly more related to theDQB1 andDQB2 genes than to other class II genes. TheDQB3 sequence mapped betweenDQA2 andDQB1, 15 kb upstream fromDQA2, by analysis of overlapping cosmid clones. This mapping was supported by the fact thatTaq I,Msp I, andBam HIDQB3 polymorphisms were perfectly correlated with theDQA2 polymorphism and not with any polymorphisms in theDR orDQ subregion, suggesting the presence of a hot spot for recombination betweenDQB3 andDQB1. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M26577.     

Primate DRB6 pseudogenes: clue to the evolutionary origin of the HLA-DR2 haplotype

The HLA-DR2 haplotype contains three \-chain encoding DRB genes and one -chain encoding DRA gene. Of the three DRB genes, two are presumably functional (HLA-DRB1 and HLA-DRB5), whereas the third (HLA-DRBV1) is a pseudogene. A pseudogene closely related to HLA-DRBVI is present in the chimpanzee (Patr-DRB6) and in the gorilla (Gogo-DRB6). We sequenced the HLA-DRBVI and Patr-DRB6 pseudogenes (all exons and most of the introns), and compared the sequence to that of the Gogo-DRB6 gene (of which only the exon sequence is available). All three pseudogenes seem to lack exon 1 and contain other deletions responsible for shifts in the translational reading frame. At least the HLA-DRBVI pseudogene, however, seems to be transcribed nevertheless. The chimpanzee pseudogene contains two inserts in intron 2, one of which is an Alu repeat belonging to the Sb subfamily, while the other remains unidentified. These inserts are lacking in the human gene. A comparison with sequences published by other investigators revealed the presence of the HLA-DRBVI pseudogene also in the DRI and DRw10 haplotypes. Measurements of genetic distances indicate DRB6 to be closely related to the DRB2 pseudogene and to the HLA-DRB4 functional gene. In humans, gorillas, and chimpanzees, the DRB6 pseudogene is associated with the same functional gene (DRB5) indicating that this linkage disequilibrium is at least six million years old and that DR2 is one of the oldest DR haplotypes in higher primates.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M77284-M77295. Address correspondence and offprint requests to: J. Klein.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Biochemistry of HLA-DRw6: evidence for seven distinct haplotypes

The DRw6 specificity, which has a frequency of 11% in the Caucasian population, cannot be positively defined, since no monospecific allo-antiserum is available. This particular status among DR specificities led us to study the DRw6 haplotypes at the molecular level. We performed 2D-PAGE analysis of HLA-DR molecules in 44 different DRw6 haplotypes. The data were obtained from six homozygous typing cells, eight families informative for the segregation of the DRw6 haplotype, and 15 unrelated donors. Five unique beta-chain electrophoretic patterns were detected, indicating the existence of five structurally distinct DRw6 beta-chains. Each haplotype expresses one or two beta-chains. The different combinations of the DR beta-chains present in a single haplotype allow to characterize seven unique DRw6 haplotypes. In contrast to what has been previously found for DR2 and DR4, there is no DR beta-chain common to all the DRw6 cells. Correlation of the biochemical data with the recent serologic (DRw13 vs DRw14) and cellular (Dw9, Dw18, Dw19) splits of the DRw6 specificity will be discussed.     

Mapping of theHox-3.1 andMyc-1.2 genes on chromosome 15 of the mouse by restriction fragment length variations

Restriction endonuclease fragment length variations (RFLV) were detected by use of the cDNA probeHox-3.1 for the homeo box-3.1 gene and also thec-myc oncogene probe for exon 2. RFLV ofHox-3.1 were found inHindIII restriction patterns, and RFLV of theMyc-1.2 gene inEcoRV patterns. From the RFLV, theHox-3.1 andMyc-1.2 genes were mapped on chromosome 15. Three-point cross test data showed that the frequency of recombination is 26.4% betweenMyc-1.2 andGpt-1, 30.2% betweenGpt-1 andGdc-1, and 9.4% betweenGdc-1 andHox-3.1. The following order of these genes is proposed,Myc-1.2—Gpt-1—Gdc-1—Hox-3.1. All laboratory strains carry theHox-3.1 ^a andMyc-1.2 ^a alleles. Among strains of wild origin,domesticus strains carry only theHox-3.1 ^a andMyc-1.2 ^a alleles, as do the laboratory strains. One strain ofbrevirostris carries theHox-3.1 ^a andMyc-1.2 ^b alleles. Other wild subspecies from Europe and Asia,M. m. musculus, M. m. castaneus, M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai carry theHox-3.1 ^b andMyc-1.2 ^b alleles.     

A possible association between basic amino acids of position 13 of DRB1 chains and autoimmune hepatitis

Fifty-one patients with autoimmune hepatitis have been studied for HLA association by conventional serology and also by modified polymerase chain reaction-restriction fragment lenght polymorphism (PCR-RFLP) genotyping.HLA-DR4 was significantly associated with autoimmmune hepatitis (46 of 51 patients, 90.2%). DNA typing of the DRB1 gene for 43 DR4-positive patients by using the PCR-RFLP technique revealed that of 43 patients, 33 had DRB1 ^* 0405 (Dw15), five had DRB1 ^* 0406 (DwKT2), four had DRB1 ^* 0403 (Dw13a), two had DRB1 ^* 0401 (Dw4), two of 43 had DRB1 ^* 0407 (Dw13b) and one had DRB1 ^* 0408 (Dw14b). Thus, there was no significant difference in Dw frequencies between DR4-positive patients and DR4-positive healthy subjects. These findings suggest that the DR4-specific sequence (Val 11 and His 13 at amino acid positions 11 and 13, respectively), but not particular Dw-associated DR4 sequence, in the first domain of the DRB1 chain contributes to susceptibility to autoimmune hepatitis among Japanese. Interestingly, all five of the DR4-negative patients had the DR2 specificity (DRB1 ¹⁵⁰² or 1601). Taken together, these results imply that the basic amino acids at position 13, which is present only on the DR2 and DR4 B1 molecules (Arg on DR2 and His on DR4), are most important for determining the predisposition to autoimmune hepatitis. Address correspondence and offprint requests to: M. Ota.     

Primate DRB genes from the DR3 and DR8 haplotypes contain ERV9 LTR elements at identical positions

The HLA-DRB genes of the human major histocompatibility complex constitute a multigene family with a varying number of DRB genes in different haplotypes. To gain further knowledge concerning the evolutionary relationship, the complete nucleotide sequence was determined for a region spanning introns 4 and 5 of the three DRB genes (DRB1*0301, DRB2 and DRB3*0101) from a DR52 haplotype and the single DRB gene (DRB1*08021) in the DR8 haplotype. These analyses identified an endogenous retroviral long terminal repeat element (ERV9 LTR3), inserted at identical positions in intron 5 of the functional DRB genes in these two haplotypes. Comparison of the nucleotide sequence from introns 4 and 5 including the ERV9 LTR elements revealed a strong similarity between the three expressed DRB genes. The DRB3*0101 and DRB1*08021 genes were most similar in this comparison. These findings provide further evidence for a separate duplication in a primordial DR52 haplotype followed by a gene contraction event in the DR8 haplotype. A homologous element was found in a chimpanzee DRB gene from a DR52 haplotype. This represents the first characterized ERV9 LTR element in a nonhuman species. The corresponding introns of the DRB genes in the DR4 haplotype contain no ERV9 LTRs. In contrast, these genes have insertions of distinct Alu repeats, implying distinct evolutionary histories of DR52 and DR53 haplotypes, respectively. Phylogenetic analyses of DRB introns from DR52, DR53, and DR8 haplotypes showed a close relationship between the DRB2 and DRB4 genes. Thus, the ancestral DR haplotype that evolved to generate the DR52 and DR53 haplotypes most likely shared a primordial common DRB gene.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X82660–X82663     

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.     

Phosphorylation-induced conformational changes in the phosphorylaseab hybrid as revealed by resolution of pyridoxal 5′-phosphate with imidazole citrate and cysteine

Summary The accessibility of pyridoxal 5′-phosphates of the phosphorylaseab hybrid to resolution by imidazole citrate and cysteine was studied and compared with that of theb anda forms. Promotion of resolution of phosphorylated forms by raising the temperature or in the presence of glycogen indicates that the resistance of phosphorylasea andab to resolution at 0°C is due rather to their tetrameric state than their phosphorylation-related active conformation. The pattern of resolution of theab hybrid was similar to that of thea and differed from that of theb forms in that it occurred at 30°C and 37°C but not at 0°C, moreover, it did not show first-order kinetics. On the other hand, inhibition of resolution by ligands binding to the nucleotide site of phosphorylase reflected an intermediate sensitivity of theab form between that of theb anda forms. We conclude that partial phosphorylation of phosphorylaseb elicits conformational change(s) in both subunits which influence the monomer-monomer interactions and resolution of pyridoxal 5′-phosphates. Resistance ofab hybrid to monomerizing agents as imidazole citrate, comparable to that of other forms, argues for its stability, ruling out its reshuffling into mixtures of phosphorylaseb anda.