Complete nucleotide sequence of a functional HLA-DP beta gene and the region between the DP beta 1 and DP alpha 1 genes: comparison of the 5'' ends of HLA class II genes.

The complete nucleotide sequence of an HLA-DP beta 1 gene and part of the adjacent DP alpha 1 gene, up to and including the signal sequence exon, were determined. The sequence of the DP beta 1 gene identified it as the DPw4 allele. The six exons of the DP beta 1 gene spanned over 11,000 bp of sequence. The arrangement of the gene was broadly analogous to genes of other class II beta chains. The beta 1 exon was flanked by introns of over 4 kb. Comparisons with published sequences of cDNA clones indicated that an alternative splice junction, at the 3' end of the gene, is used in at least one allele. Variation in choice of splice junction indicates an additional mechanism for allelic variation in class II genes. The sequence also indicated that the DP beta 1 and DP alpha 1 genes are separated by only 2 kb at their 5' ends. Comparison of the 5' ends of the DP alpha 1 and beta 1 genes with other class II sequences, including the DZ alpha gene, showed conservation of several blocks of sequences thought to be involved in control of expression. Some areas of the introns were partially conserved in the DQ beta gene, and several other intron sequences were homologous to sequences found in other unrelated genes.     

Construction and characterization of cDNA clones encoding the 5' end of the chicken pro alpha 1(I) collagen mRNA

As a first step in isolating the 5' end of the chicken pro alpha 1(I) collagen gene, we constructed cDNA clones complementary to the 5' end of the pro alpha 1(I) mRNA using synthetic oligodeoxynucleotides complementary to a conserved region within the N-terminal telopeptide as primers. cDNA clones corresponding to the 5'-untranslated region, signal peptide, N-propeptide and telopeptide were identified based on homology with the human pro alpha 1(I) collagen protein sequence, and on hybridization to pro alpha 1(I) mRNA on Northern blots. A comparison of the nucleotide sequence of these clones with the sequence of the 5' end of the pro alpha 2(I) collagen mRNA confirms that there is 84% homology in a 49-bp region surrounding the translation start point, and shows that there is 70% homology in the nucleotide sequences encoding the N-propeptide triple helical region of the two type-I collagen chains.     

Mechanisms of divergence and convergence of the human immunoglobulin alpha 1 and alpha 2 constant region gene sequences

Nucleotide sequences of the human alpha 1 and two allelic alpha 2 immunoglobulin heavy chain constant region genes are presented. The genes contain three exons, each encoding a single constant region protein domain. The protein hinge region is encoded at the 5' end of the second exon, and the rapid evolutionary changes in length of the hinge correspond to duplications or deletions within the hinge-coding region, probably facilitated by repeats in the DNA sequence. Alignment of the alpha 1 and alpha 2 gene sequences reveals an unusual coupled deletion-duplication in the 5'-flanking region, which can be explained in terms of a slipped-strand mispairing model. Comparison of nucleotide sequences of the alpha 1 gene and two alleles of the alpha 2 gene indicates a localized transfer of genetic information from the 3' end of the alpha 1 gene to one of the alpha 2 alleles, probably by a gene conversion. At one end of the region within which conversion apparently occurred, there is a 40 bp sequence of the type that can form Z-DNA.     

Structure and expression of class II alpha genes in miniature swine.

Two overlapping genomic clones corresponding to the swine DRA class II gene were isolated and characterized. Restriction mapping and partial sequence data of the exon-containing fragments allowed identification and orientation of the five exons encoding the alpha chain. Two full length cDNA clones corresponding to the transcribed DRA gene from two different haplotypes of the swine MHC were sequenced. Nucleotide sequence alignments revealed that the two swine DRA cDNA were very similar and closely related to the human DRA equivalent. An additional glycosylation site, compared with those of human DRA, was found in the second external domain of the protein. Northern analyses showed that porcine DRA and DQA genes were the only two class II alpha genes expressed in the spleen, despite the presence of DPA and DZA genes in the genome. In addition to transfected cells expressing homologous pairs of alpha and beta chains from SLA-DR, stable transfectants expressing nonhomologous pairs of alpha and beta chains from DR and DQ loci were obtained, suggesting that such associations may contribute to the functional heterogeneity of class II products.     

Organization of the murine Ig-related lambda 5 gene transcribed selectively in pre-B lymphocytes.

lambda 5 is an immunoglobulin lambda light chain-related gene which is selectively transcribed in murine pre-B lymphocytes to yield a 1.2 kb poly(A)+ mRNA. Comparison of the nucleotide sequence of a 1 kb cDNA clone with the sequence of a genomic clone isolated from 70Z/3 murine pre-B lymphoma cells shows lambda 5 is composed of three exons spanning a 3.75 kb DNA segment. Conserved splice signal sequences at all exon/intron boundaries and the presence of a long open reading frame indicate that a functional mRNA molecule can be made. Exon I contains a cap-site and a potential ATG start codon as well as sequences encoding a signal peptide. This gene could encode a lambda 5 protein of 209 amino acids which has, however, not yet been identified. The 3' portion of exon II and all of exon III shows strong sequence homologies to J lambda L and C lambda L exons. Homology to the lambda L chain genes is lost in the 5' portion of exon II and throughout exon I. In exon I short homologies to leader sequences and to VH framework 1 sequences are seen.     

Molecular genetics of human erythrocyte MiIII and MiVI glycophorins. Use of a pseudoexon in construction of two delta-alpha-delta hybrid genes resulting in antigenic diversification

Human glycophorins alpha and delta (or A and B) specify the MNSs blood group antigens; they exhibit considerable structural variation among populations. We show that two variant phenotypes of Miltenberger class III and VI are encoded by similar hybrid glycophorin genes in a delta-alpha-delta arrangement. Restriction mapping identified altered fragments unique to the MiIII and MiVI genes. Genomic sequences spanning exons 2 to 4 of the two genes were obtained by allele-specific polymerase chain reaction. Restriction analysis and direct sequencing of the amplified DNA revealed that MiIII and MiVI genes are identical to the delta gene except that, in both, an internal segment of the delta gene has been replaced by its homologous counterpart of the alpha gene, resulting in a delta-alpha-delta hybrid structure. In the process of hybrid formation a portion of alpha exon 3 and intron 3, that carries a functional 5' splicing signal, has been fused to an exon-like sequence in the delta gene that retains a 3' but lacks a 5' splicing signal. These rearrangements created a composite exon resulting in the expression of the ordinarily unexpressed delta gene sequence and conferred the hybrid proteins with new antigenic specificities. The expression of this sequence in MiIII glycophorin is directly demonstrated by protein sequencing. MiIII and MiVI genes differ in the location of upstream (delta-alpha) and downstream (alpha-delta) breakpoints and in the length of sequence replacement. The delta-alpha breakpoints of the two genes occur at different locations within a 35-base pair sequence of exon 3 that is clustered with multiple inverted repeats, whereas the alpha-delta breakpoints reside downstream in two dissimilar blocks of sequences of intron 3. The minimal length of the delta gene sequence that has been replaced by the alpha gene is 55 base pairs in the MiIII gene and 131 base pairs in the MiVI gene. Such segmental DNA transfers may have proceeded unidirectionally through the mechanisms of gene conversion.     

Class II genes of the human major histocompatibility complex. The DO beta gene is a divergent member of the class II beta gene family

A novel class II beta chain gene is described. This gene, tentatively called DO beta, displays considerably less polymorphism than beta genes of the DP, DQ, and DR loci. The nucleotide sequence of the DO beta gene is strikingly similar to that of the previously identified murine A beta 2 gene. The DO beta gene displays the same exon/intron organization as other beta genes although the fifth exon and the translated portion of the sixth exon are longer than in other genes. A striking feature of the amino acid sequence deduced from the DO beta gene sequence is the pronounced hydrophobicity of the NH2-terminal region. This feature distinguishes the putative DO beta chain from other class II beta chains and raises the possibility that DO beta chains may interact with an alpha chain that is structurally different from those of the DP, DQ, and DR loci. It further suggests that the putative DO molecule may have a function different from those of other class II antigens.     

Class II genes of the human major histocompatibility complex. Comparisons of the DQ and DX alpha and beta genes

The human major histocompatibility complex, HLA, contains the genes of several class II molecules. We present here the molecular maps of the DQ and DX subregions and analyze the sequences of the polymorphic DQ alpha and DQ beta genes as well as the DX alpha and DX beta genes. The DQ alpha and DQ beta genes are oriented in opposite directions, approximately 12 kilobases apart. The DX alpha and DX beta genes are similarly oriented about 8 kilobases. The exon-intron organizations of the DQ alpha and DX alpha genes are analogous to those of other class II alpha genes. Comparison of the DQ alpha gene sequence to three DQ alpha cDNA clones shows that amino acid replacements are predominantly located between residues 45 and 80 in the amino-terminal domain. Analysis of the frequency of silent and replacement substitutions indicates that there is little selection against replacements in DQ alpha first domains. The exons encoding the second domains of DQ alpha and DX alpha are virtually identical, suggesting that a gene conversion event has occurred between these genes. The DX beta gene is very similar to the DQ beta gene but differs in the cytoplasmic portion. The DX beta gene contains a separate exon of 24 nucleotides encoding the core of the cytoplasmic tail. This exon is not expressed in the DQ beta genes due to a nonfunctional splice junction. Comparison of the number of nucleotide substitutions in the DQ beta first and second domain exons suggests that little or no phenotypic selection acts on the first domain whereas the second domain is under strong selection.     

The human alpha 2(XI) collagen (COL11A2) chain. Molecular cloning of cDNA and genomic DNA reveals characteristics of a fibrillar collagen with differences in genomic organization

We have isolated three overlapping cDNA clones encoding the pro alpha 2(XI) collagen chain from a human chondrocyte cDNA library. Together, the cDNAs code for 257 uninterrupted Gly-X-Y triplets (almost 80% of the triple helical domain) and about 200 amino acid residues of the carboxyl telopeptide and carboxyl propeptide. The identification of the clones as pro alpha 2(XI) cDNAs was based on the complete identity between the amino acid sequences of three tryptic peptides derived from human alpha 2(XI) collagen and the cDNA-derived sequence. We have also sequenced six exons within a human genomic alpha 2(XI) cosmid clone. This sequence shows that although type XI collagen belongs to the fibril-forming class of collagens, there are substantial differences in exon sizes at the 3' end of the gene when comparing the alpha 2(XI) gene with those of human types I, II, and III collagens. Finally, pro alpha 2(XI) cDNA has been used as a probe to determine the location of the gene by in situ hybridization of chromosome spreads. The results demonstrate that the gene is located close to the region p212 on chromosome 6. Northern blot analysis shows that the gene is expressed in cartilage but not in adult liver, skin, and tendon.