Structure,expression, chromosomal location and product of the gene encoding ADH1 inPetunia

A genomic clone for an alcohol dehydrogenase (Adh) gene has been isolated fromPetunia hybrida cv. V30 by screening aPetunia genomic library with a maizeAdh1 probe. A combination of RFLP and allozyme segregation data failed to demonstrate which of twoAdh loci, both of which map to chromosome 4, was the source of the cloned gene. The product of the cloned genes has been identified unequivocally by a transient expression assay inPetunia protoplasts. We have designated this genePetunia Adh1. The expression of this gene is tightly regulated in the developing anther, where its gene product is the predominant ADH isozyme. It is anaerobically inducible in roots, stems and leaves of seedlings. The induction of enzyme activity is correlated with induction ofAdh1 mRNA.     

Structure and chromosomal location of the human gene encoding cartilage matrix protein

Cartilage matrix protein (CMP) is a major component of the extracellular matrix of nonarticular cartilage. The structure and chromosomal location of the human gene encoding CMP was determined by molecular cloning analysis. We used a partial chicken CMP cDNA probe to isolate three overlapping human genomic clones. From one of these clones, a probe containing 2 human CMP exons was isolated and used to map the gene to chromosome 1p35 and to screen a human retina cDNA library. Two overlapping cDNA clones were isolated. The predicted protein sequence of 496 amino acids includes a 22-residue signal peptide and a 474-residue mature protein of Mr 51,344. The human CMP gene and polypeptide are strikingly similar to the chicken CMP gene and polypeptide. Human CMP is 79% identical to chicken CMP and contains two homologous domains separated by an epidermal growth factor-like domain. One potential N-glycosylation site is conserved between the two species. The human CMP gene spans 12 kilobase pairs with 8 exons and 7 introns which are similar in size to those of the chicken CMP gene. Both RNA splice junctions of intron G in the human and chicken CMP genes are nonconforming to the consensus splice sequences. This suggests that the CMP gene utilizes a new RNA splicing mechanism.     

Genomic organization and chromosomal location of the human gene encoding the B-lymphocyte activation antigen B7

The human B lymphocyte activation antigen B7 provides regulatory signals for T lymphocytes as a consequence of binding to its ligands CD28 and CTLA-4. The cDNA for B7 has previously been isolated and predicted to encode a type I membrane protein. The predicted polypeptide has a secretory signal peptide followed by two contiguous Ig-like domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Here we report the exon-intron genomic organization of human B7 and the chromosomal location. The gene has six exons that span approximately 32 kilobases of DNA. Exon 1 is not translated and the second exon contains the initiation ATG codon and encodes a predicted signal peptide. This gene structure is characteristic for several eukaryotic genes with tissue-specific expression. The third and fourth exons correspond to two Ig-like domains whereas the fifth and sixth exons encode respectively the trans-membrane portion and the cytoplasmic tail. This close relationship between exons and functional domains is a characteristic feature of genes of the Ig superfamily. Cell surface expression of the B7 gene product has previously been mapped to human chromosome 12 by antibody reactivity with the B7-specific monoclonal antibody BB-1. We here demonstrate that theB7 gene is located to theq21-qter region of chromosome 3 by DNA blot analysis of human × rodent somatic cell hybrids.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M83071-M83075, M83077. Address correspondence and offprint requests to: B. Dupont, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue (Room S709), New York, NY 10021, USA.     

Structure, expression and chromosomal location of the Oct-4 gene.

The map position of Oct-4 on mouse chromosome 17 is between Q and T regions in the Major Histocompatibility Complex (MHC), and it is physically located within 35 kb of a class I gene. Several Oct-4-related genes are present in the murine genome; one of them maps to chromosome 9. The genomic structure and sequence of Oct-4 determined in t-haplotypes reveals five exons, and shows no significant changes in the t12 mutant haplotype making it unlikely that Oct-4 and the t12 early embryonic lethal are the same gene. By in situ hybridization, detectable onset of zygotic Oct-4 expression does not occur until compaction begins at 8-cells, suggesting that there might be other regulatory factors responsible for initiating Oct-4 expression.     

Structure and chromosomal location of the human granulin gene.

Granulins are a family of cysteine rich polypeptides some of which have growth modulatory activity. We showed previously that the granulins are encoded within the same precursor consisting of seven granulin domains arranged in tandem. Here we report the chromosomal location and structural organization of the protein coding region of the granulin gene. The granulin gene was assigned to chromosome 17 using DNA from human-hamster somatic cell hybrids. The protein-coding region of the granulin gene was shown to comprise 12 exons covering about 3700 bp. Each tandem granulin repeat is encoded by two non-equivalent exons, a configuration unique to the granulins that would permit the formation of hybrid granulin-like proteins by alternate splicing.     

Structural organization and chromosomal assignment of the gene encoding endothelin

Endothelin is a 21-amino acid vasoconstrictor synthesized and secreted by vascular endothelial cells. The human peptide is derived from a 212-amino acid precursor, preproendothelin. A nearly full length clone containing DNA complementary to human preproendothelin mRNA was isolated, and its nucleotide sequence was determined. Using this cDNA as a probe, the genomic organization of the human endothelin gene was determined and the promoter region delineated. The gene contains five exons and four intervening sequences. Nucleotide sequences encoding endothelin are contained within the second exon, and the third exon specifies a portion of preproendothelin that is homologous to endothelin. The second and third exons may represent descendants of a common progenitor exon. The 3'-untranslated portion of the gene contains a 250-base pair region that is highly conserved between human and porcine genomes and may have an important role in endothelin mRNA stability. On the basis of DNA isolated from human-mouse somatic hybrid cell lines, the endothelin gene was assigned to human chromosome 6.     

Cloning and characterization of a gene encoding wheat starch synthase I

 A cDNA clone, and a corresponding genomic DNA clone, containing full-length sequences encoding wheat starch synthase I, were isolated from a cDNA library of hexaploid wheat (Triticum aestivum) and a genomic DNA library of Triticum tauschii, respectively. The entire sequence of the starch synthase-I cDNA (wSSI-cDNA) is 2591 bp, and it encodes a polypeptide of 647 amino-acid residues that shows 81% and 61% identity to the amino-acid sequences of SSI-type starch synthases from rice and potato, respectively. In addition, the putative N-terminal amino-acid sequence of the encoded protein is identical to that determined for the N-terminal region of the 75-kDa starch synthase present in the starch granule of hexaploid wheat. Two prominent starch synthase activities were demonstrated to be present in the soluble fraction of wheat endosperm by activity staining of the non-denaturing PAGE gels. The most anodal band (wheat SSI) shows the highest staining intensity and results from the activity of a 75-kDa protein. The wheat SSI mRNA is expressed in the endosperm during the early to mid stages of wheat grain development but was not detected by Northern blotting in other tissues from the wheat plant. The gene encoding the wheat SSI (SsI-D1) consists of 15 exons and 14 introns, similar to the structure of the rice starch synthase-I gene. While the exons of wheat and rice are virtually identical in length, the wheat SsI-D1 gene has longer sequences in introns 1, 2, 4 and 10, and shorter sequences in introns 6, 11 and 14, than the corresponding rice gene. Received: 5 June 1998 / Accepted: 29 September 1998     

Molecular analysis of the gene encoding a rice starch branching enzyme

Summary The sequence of a rice gene encoding a starch branching enzyme (sbe1) shows extreme divergence from that of the rice gene, that is homologous to bacterial glycogen branching enzyme (sbe2). sbe1 is expressed abundantly and specifically in developing seeds and maximally in the middle stages of seed development. This expression pattern completely coincides with that of the waxy gene, which encodes a granule-bound starch synthase. Three G-box motifs and consensus promoter sequences are present in the 5 flanking region of sbe1. It encodes a putative transit peptide, which is required for transport into the amyloplast. A 2.2 kb intron (intron 2) precedes the border between the regions encoding the transit peptide and the mature protein, and contains a high G/C content with several repeated sequences in its 5 half. Although only a single copy of sbe1 is present in the rice genome, Southern analysis using intron 2 as a probe indicates the presence of several homologous sequences in the rice genome, suggesting that this large intron and also the transit peptide coding region may be acquired from another portion of the genome by duplication and insertion of the sequence into the gene.     

Human cellular retinol-binding protein gene organization and chromosomal location

The gene encoding the human cellular retinol-binding protein (CRBP) has been isolated from genomic libraries and its structure determined. Only one copy of the gene is present in the human genome. We have located the CRBP gene to segment 3p11-3qter on human chromosome 3 using hybridizations to mouse-human, rat-human and hamster-human cell hybrids. The gene harbors four exons encoding 24, 59, 33, and 16 amino acid residues respectively. The second intervening sequence alone occupies 19 kb of the 21 kb of the CRBP gene. The nucleotide sequence of the gene has been determined with the exception of the second intron. The positions of the introns agree with those in the rat CRBPII, the rat liver fatty-acid-binding protein and the mouse adipose P2 protein genes encoding molecules belonging to the same protein family as CRBP. In contrast to the other sequenced members of this family the promoter of the CRBP gene resembles those found in the 'housekeeping' genes in that it is (G + C)-rich, contains multiple copies of the CCGCCC sequence and lacks TATA box. A 9-bp homology containing the core sequence of the simian virus 40 enhancer repeat was found in the 5' upstream region. A genomic Southern blot probed with CRBP cDNA revealed hybridizing bands in restricted chicken and frog DNA.     

Identification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds.

Three forms of soluble starch synthase were resolved by anion-exchange chromatography of soluble extracts from immature rice (Oryza sativa L.) seeds, and each of these forms was further purified by affinity chromatograph. The 55-, 57-, and 57-kD proteins in the three preparations were identified as candidates for soluble starch synthase by western blot analysis using an antiserum against rice granule-bound starch synthase. It is interesting that the amino-terminal amino acid sequence was identical among the three proteins, except that the 55-kD protein lacked eight amino acids at the amino terminus. Thus, these three proteins are products of the same gene. The cDNA clones coding for this protein have been isolated from an immature rice seed library in lambda gt11 using synthetic oligonucleotides as probes. The deduced amino acid sequence of this protein contains a lysine-X-glycine-glycine consensus sequence for the ADP-glucose-binding site of starch and glycogen synthases. Therefore, we conclude that this protein corresponds to a form of soluble starch synthase in immature rice seeds. The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino terminus. The mature form of soluble starch synthase shares a significant but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase. However, several regions, including the substrate-binding site, are highly conserved among these three enzymes. Blot hybridization analysis demonstrates that the gene encoding soluble starch synthase is a single-copy gene in the rice genome and is expressed in both leaves and immature seeds. These results suggest that soluble and granule-bound starch synthases play distinct roles in starch biosynthesis of plant.     

Structure and mapping of the gene encoding mouse high affinity Fc gamma RI and chromosomal location of the human Fc gamma RI gene.

We describe the isolation and characterization of the gene encoding the mouse high affinity Fc receptor Fc gamma RI. Using a mouse cDNA Fc gamma RI probe four unique overlapping genomic clones were isolated and were found to encode the entire 9 kb of the mouse Fc gamma RI gene. Sequence analysis of the gene showed that six exons account for the entire Fc gamma RI cDNA sequences including the 5'- and 3'-untranslated sequences. The first and second exons encode the signal peptide; exons 3, 4, and 5 encode the extracellular Ig binding domains; and exon 6 encodes the transmembrane domain, the cytoplasmic region, and the entire 3'-untranslated sequence. This exon pattern is similar to Fc gamma RIII and Fc epsilon RI but differs from the related Fc gamma RII gene which contains 10 exons and encodes the b1 and b2 Fc gamma RII. Southern blot analysis had shown that the mouse Fc gamma RI gene is a single copy gene with no RFLP in inbred strains of mice, but analysis of an intersubspecies backcross of mice showed that unlike other mouse FcR genes which are on mouse chromosome 1 the locus encoding Fc gamma RI, termed Fcg1, is located on chromosome 3. Interestingly, the Fcg1 locus is located near the end of a region with known linkage homology to human chromosome 1. Analysis of human x rodent somatic cell hybrid cell lines indicates that the human FCG1 locus encoding the human Fc gamma RI maps to chromosome I and therefore possibly linked to other FcR genes on this chromosome. These results suggest that the linkage relationships among these genes in the human genome are not preserved in the mouse.