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Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (mHS) is the first enzyme of ketogenesis, whereas the cytoplasmic HS isozyme (cHS) mediates an early step in cholesterol synthesis. We here report the sequence of human and mouse liver mHS cDNAs, the sequence of a HS-like cDNA from Caenorhabditis elegans, the structure of a partial human mHS genomic clone, and the mapping of the human mHS gene to chromosome 1p12-p13. The nucleotide sequence of the human mHS cDNA encodes a mature mHS peptide of 471 residues, with a mean amino acid identity of 66.5% with cHS from mammals and chicken. Comparative analysis of all known mHS and cHS protein and DNA sequences shows a high degree of conservation near the N-terminus that decreases progressively toward the C-terminus and suggests that the two isozymes arose from a common ancestor gene 400-900 million years ago. Comparison of the gene structure of mHS and cHS is also consistent with a recent duplication event. We hypothesize that the physiologic result of the HS gene duplication was the appearance of HS within the mitochondria around the time of emergence of early vertebrates, which linked preexisting pathways of beta oxidation and leucine catabolism and created the HMG CoA pathway of ketogenesis, thus providing a lipid-derived energy source for the vertebrate brain.

The 51-kDa flavoprotein subunit of mitochondrial NADH:ubiquinone oxidoreductase (Complex I) [NADH dehydrogenase (ubiquinone), flavoprotein 1 (51 kDa); EC 1.6.5.3] plays an important role in the formation of the NADH-binding site and is believed to be the principal site of entry for electrons donated by NADH into the respiratory chain. Human cDNA fragments of the 51-kDa protein were generated by polymerase chain reaction and used to localize the gene (NDUFV1) for this subunit to 11q13 by two separate techniques. This region of the human genome is strongly implicated in a number of different forms of cancer.

A family of proinflammatory cytokines sharing several structural features has been described and includes, for example, interleukin-8, monocyte chemoattractant protein-1, and melanocyte growth stimulatory activity. Recently, the receptors for interleukin-8 have been isolated and found to belong to the seven-transmembrane domain class of G protein-coupled receptors. As other members of this cytokine family likely interact with similar receptors, the polymerase chain reaction was employed to isolate related receptors from human peripheral blood adherent cells. Degenerate oligonucleotide primers based on the rabbit interleukin-8 receptor sequence were used. The corresponding full-length cDNA was isolated from a human spleen cDNA library. The predicted protein sequence of this clone, designated pBE1.3, was 93% identical to that of a cDNA isolated from bovine locus coeruleus, which apparently encodes a neuropeptide Y receptor, and also shows similarity with the interleukin-8 receptor and the human cytomegalovirus US28 sequences. The gene, designated D2S201E was localized to human chromosome 2q21. By Northern blotting, transcripts hybridizing to this cDNA were present in a variety of tissues and cells, including those of hemopoietic origin.

3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL) is a homodimeric mitochondrial matrix enzyme that catalyzes the last step of ketogenesis. Using a human HL cDNA as a probe, we isolated a 1.4-kb mouse HL cDNA (HLM) from a mouse liver library and extended the sequence in the 5' direction, using RACE PCR to include the complete coding sequence. The nucleotide sequence of the mouse HL coding region is 85.7% identical to human HL, and 52.6% to Ps. mevalonii HL. Peptide identities of 87.4% and 54.3% respectively were observed. Southern analysis of 29 strains of laboratory mice and of Mus spretus revealed a total of about 25 kb of hybridizing fragments and three polymorphic fragments in both EcoRI and Hin-dIII digestions. The mouse HL locus (Hmgcl) was localized on Chromosome (Chr) 4: Pmv-19-12.6 +/- 3.6 cM-Hmgcl-7.3 +/- 2.3 cM-Xmv-8-1.5 +/- 1.0 cM-Gpd-1. The human HL locus (HMGCL) was mapped to distal Chr 1p by analysis of a human-hamster hybrid cell panel and by in situ hybridization.

The genetic defect underlying Huntington's disease (HD) has been mapped to 4p16.3. Refined localization using recombinant HD chromosome analysis and allelic association analyses have identified two distinct candidate regions. Using a cDNA hybrid selection procedure we have cloned the gene for alpha-adducin, a subunit of a cytoskeletal protein crucial for spectrin-actin membrane plasticity. This gene maps to the proximal 2.2 Mb candidate region within 20 kb of D4S95. Alleles of markers at this locus have been shown to exhibit significant linkage disequilibrium with HD. A 4 kb alpha-adducin transcript was identified which is abundantly expressed in the caudate nucleus, the site of major neuronal loss in HD. Sequencing of the brain alpha-adducin cDNA from two HD patients and an age-matched control did not detect any sequence alterations specific to HD. However, we identified in brain cDNA of both patients and control samples, two alternately spliced brain exons, not previously described in the erythrocyte cDNA. A 93 bp exon is inserted in frame between codon 471 and 472 while a 34 bp exon inserted within codon 621 disrupts the frame and introduces a stop codon after 11 novel amino acids. The mapping of the adducin gene adjacent to D4S95 and its pattern of expression, as well as its potential for distinct alternately spliced variants, reinforces the necessity to accurately assess the role of the expression of this gene in the pathogenesis of HD.

The biosynthesis of protein-bound complex N-glycans in mammals requires a series of covalent modifications governed by a large number of specific glycosyltransferases and glycosidases. The addition of oligosaccharide to an asparagine residue on a nascent polypeptide chain begins in the endoplasmic reticulum. Oligosaccharide processing continues in the Golgi apparatus to produce a diversity of glycan structures. UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) is a key enzyme in the process because it is essential for the conversion of high-mannose N-glycans to complex and hybrid N-glycans. We have isolated the mouse gene encoding GlcNAc-TI (Mgat-1) from a genomic DNA library. The mouse sequence is highly conserved with respect to the human and rabbit homologs and exists as a single protein-encoding exon. Mgat-1 was mapped to mouse Chromosome 11, closely linked to the gene encoding interleukin-3 by the analysis of multilocus interspecies backcrosses. RNA analyses of Mgat-1 expression levels revealed significant variation among normal tissues and cells.

As part of the search for the Huntington disease (HD) gene we have cloned and sequenced 34 kb of genomic DNA containing the full-length gene for the beta-subunit of the human cGMP phosphodiesterase (beta-cGMP PDE). This gene is localized to 4p16.3 about 700 kb proximal to the 4p telomere and represents the most telomeric gene characterized on 4p to date. We show that this gene is comprised of 22 exons spanning approximately 43 kb of genomic DNA. We also provide 400 bp immediately 5' to the putative initiator methionine and 700 bp of 3' flanking sequences. Northern blot analysis of several human tissues revealed a highly abundant 3.5 kb transcript and a minor signal of 4.5 kb in retinal tissue. Alignment of the deduced amino acid sequence to the previously identified beta-subunits of the cGMP PDEs of mouse and cow demonstrates highly significant similarities and, therefore, confirms the identity of the cloned gene. A defect in the beta-subunit of the cGMP PDE gene has been shown recently to be the cause for the retinal degeneration in the rd mouse. The cloning of the human homolog and the knowledge of its genomic organization with exon/intron boundaries will allow rapid assessment of the role of this gene in the causation of human retinopathies.