Report of the committee on the genetic constitution of chromosome 6

We have determined the localization and orientation of two genetic probes within the human major histocompatibility complex by chromosomal in situ hybridization.Our data indicate that a cloned genomic probe cross-hybridizing to HLA-A, -B, and -C heavy chain loci is homologous to sequences located on chromosome 6 at band p21.3 while a subclone of the genomic HLA-DR a-chain gene corresponding to the nonpolymorphic p34 protein is homologous to sequences in band 6p21.1. Our data suggest that this technique may permit the estimation of map distances between linked gene loci, assuming a uniform frequency of map units in the human genome. The relative positions of these genes was confirmed in a mother and son carrying a chromosome rearrangement involving 6p and 14p in which the sequences hybridizing to a DR a-chain genomic clone were found at the distal end of the 6p-chromosome [der(6)] while the sequences hybridizing to the HLA-A, -B, -C a-chain probe were found in the 14p+ chromosome [der(14)].The major histocompatibility complex (MHC) in humans is a cluster of at least three gene families encoding cell surface glycoproteins-i.e., genes for class I (HLA-A, -B, and -C) and class II (HLA-DR) antigens in addition to class III genes (complement factors)-and spans 2-3 x 103 kilobase pairs (kb) of DNA (1, 2). Genetic mapping by somatic cell hybridization (3-7) and by the segregation of unique chromosomal rearrangements involving the MHC in families (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) has permitted localization of the serologically polymorphic 45,000-dalton glycoproteins of the class I genes (HLA-A, -B, and -C) to the short arm of human chromosome 6, with the shortest region of overlap being the 6p21 band (7,17). Associated with the polymorphic glycoproteins of the HLA-A, -B, and -C antigens is a nonglycosylated 12,000-dalton invariant chain, 82-microglobulin, that has been assigned to human chromosome 15 (20,21). The class II genes, or the immune response genes, consist of heterodimers composed of a 34,000-dalton (HLA-DR a) glycoprotein for which no electrophoretic polymorphisms have been demonstrated and a polymorphic 29,000-dalton (HLA-DR (3) glycopeptide (21)(22)(23)(24)(25). Polymorphism of the HLA-DR , gene has permitted a regional assignment of this locus to chromosome 6 proximal to 6p22.4 by a family study (15).Recently, the HLA-DR a-chain locus has been mapped by using somatic cell hybrids (26) and, furthermore, this assignment has been refined to the region 6p2105-6p23 (27) by study of genomic blotting patterns in cell lines containing individual chromosomal deletions of the short arm of chromosome 6. Data on recombination within the HLA complex and with closely linked markers such as glyoxylase 1 (GLO 1) have permitted inferences to be drawn about the order and map distances between marker loci on the proximal portion of the short arm of chromosome 6, as follows: GLO 1-4 centimorgans (cM)-HLA-DR-0.3 cM-BF, C4B, C4A, C2-0.7 cM-HLA-B-0.1 cM-HLA-C-0.7 cM-HLA-A (16-18).Improvements in chromosomal i...

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

Orientation of loci within the human major histocompatibility complex by chromosomal in situ hybridization.

Kirsch¹,

Nance²,

Evans³

et al. 1984

“…The genes coding for the complement components C2, factor B, and two classes of C4 (C4A and C4B) lie between HLA-B and HLA-D in the major histocompatibility complex (HLA) in man (1). These genes have been mapped relative to each other, showing that those for C2 and factor B lie less than 1 kilobase (kb) apart and are separated from the C4A gene by about 30 kb (2).…”

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confidence: 99%

Mapping of steroid 21-hydroxylase genes adjacent to complement component C4 genes in HLA, the major histocompatibility complex in man.

Carroll¹,

Campbell²,

Porter³

1985

331

108

The genes for four components (C) of complement in the human major histocompatibility complex (HLA) have been aligned previously in a series of overlapping cosmid cloned inserts. Those inserts, which contained the two C4 genes C4A and C4B, hybridized with human adrenal mRNA, indicating that they contain a gene expressed in the adrenal. The mRNA fraction of 2.4 kilobases (kb) hybridizes with genomic DNA of 4.5 kb, which is duplicated and lies about 1.5 kb 3' of both the C4A and the C4B complement genes. Sequencing of a 430-base section and comparison with the published cDNA sequence of bovine cytochrome P-450 21-hydroxylase, peptide sequences of porcine 21-hydroxylase, and a cDNA sequence of a rat liver cytochrome P-450 identified the gene as coding for human steroid 21-hydroxylase [steroid,hydrogen-donor:oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10]. Mapping of the gene was helped by use of a synthetic oligonudeotide based on the bovine cDNA sequence.The genes coding for the complement components C2, factor B, and two classes of C4 (C4A and C4B) lie between HLA-B and HLA-D in the major histocompatibility complex (HLA) in man (1). These genes have been mapped relative to each other, showing that those for C2 and factor B lie less than 1 kilobase (kb) apart and are separated from the C4A gene by about 30 kb (2). The C4B gene is about 10 kb from the C4A gene and, on a chromosome from one individual, a second C4B gene was found 10 kb further away (3).The most common form of congenital adrenal hyperplasia is due to a defect of steroidogenesis in the adrenal in which the enzyme steroid 21-hydroxylase [steroid,hydrogen-donor:oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10] is wholly or partly missing or inactive (4). This inherited defect is closely linked to the HLA complex (5). The 21-hydroxylase enzyme is a cytochrome P-450, and a cDNA clone coding for it has been obtained from bovine adrenal mRNA (6). The derived amino acid sequence showed strong homology with cystine-containing peptide sequences of porcine 21-hydroxylase (7), and there was limited homology between the cDNA sequence of the bovine 21-hydroxylase and of a rat liver cytochrome P-450 (8).To extend the characterization of the section of the HLA complex containing the complement genes, we tested the ability of cosmids (cos-containing plasmids) and their subcloned fragments to hybridize with human adrenal mRNA. This identified the position of the 21-hydroxylase genes in HLA, and use of a synthetic oligonucleotide based on the known cDNA and amino acid sequences helped to define the position of the genes. MATERIALS AND METHODSPreparation and Analysis of Genomic DNA. Genomic DNA was prepared from whole blood by the procedure of Bell et al. (9). For restriction enzyme analysis of DNA, 8-10 ,ug of genomic DNA was digested with one or two restriction endonucleases (5 units/,ug of genomic DNA) according to the manufacturer's instructions (New England Biolabs). DNA fragments were separated by electrophoresis in 0.7% agarose gels and t...

“…Starting from these clones, we isolated and linked by overlap-hybridization methods a number of S-region cosmids. Several new genes could be expected on these cosmids, primarily those anticipated from analogy with the major histocompatibility complex of other mammals (11). However, the structural genes for the complement components C2 and factor B (see ref.…”

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confidence: 99%

Liver mRNA probes disclose two cytochrome P-450 genes duplicated in tandem with the complement C4 loci of the mouse H-2S region.

Amor¹,

Tosi²,

Duponchel³

et al. 1985

A search for uncharacterized genes of the S region of the murineH-2 major histocompatibility complex was undertaken; a series of cosmid clones previously aligned by overlap hybridizations were used as radiolabeled probes. Sequences hybridizing with liver poly(A)+ RNA were found within a cosmid covering a region 3' to the C4-Slp gene (the gene encoding the hemolytically inactive isoform of the fourth component of serum complement). Radiolabeled, short cDNA complementary to liver poly(A)+ RNA was used to establish the transcriptional polarity of the newly detected gene and to define fragments containing its 3' end. DNA sequence analyses and comparisons with porcine peptides established that the gene encodes the enzyme steroid 21-hydroxylase (EC 1.14.99.10), a cytochrome P-450 often referred to as P-450(C21), whose major site of expression is the adrenal gland. Two copies of the P-450(C21) gene, very similar yet distinguishable by restriction endonuclease analysis, were found individually associated with C4 and C4-Slp, genes that encode isoforms of mouse fourth component of complement. One of the P450(C21) genes is coamplified with C4-Slp in H-2 7, a haplotype carrying a rare elongation of the S region. Comparisons with other members of the P450 gene family show that the P-450(C21) genes encode peptides of extraordinary evolutionary conservation. The detection of a liver transcript of P-450(C21) raises the issue of the specific metabolic role of this enzyme in this organ and may have implications for the interpretation of human congenital adrenal hyperplasia.The S region of the murine H-2 major histocompatibility complex is commonly viewed as composed of loci related to the complement system, or class III genes. Although it represents the largest genetic segment (0.4 centimorgan) of the H-2 complex, surprisingly few marker genes have been definitely mapped in this region.To extend the functional characterization of this H-2 segment, we sought coding DNA sequences in cloned genomic fragments by using mRNA hybridization and overlapping cosmid clones. We now report the finding of DNA sequences that hybridize with a 20S liver mRNA and that lie immediately adjacent to the gene (C4) encoding the fourth component of complement (C4) and to the gene (C4-Slp) encoding the nonhemolytic isoform of C4, sex-limited protein (Slp). By sequencing genomic DNA, we have established that these sequences correspond to two copies of the gene encoding steroid 21-hydroxylase [21-OHase; steroid 21-monooxygenase; steroid,hydrogen-donor:oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10], a member of the cytochrome P-450 isozyme superfamily (1). MATERIALS AND METHODSPreparation of Liver DNA and RNA. Genomic DNA was isolated (2) from liver nuclei prepared by the citric acid method (3). RNA was isolated essentially as described (4) and precipitated in 2.7 M lithium chloride. The polyadenylylated RNA fraction was bound to poly(U)-Sephadex (Bethesda Research Laboratories) according to the instructions of the manufacturer but was elut...