Novel restriction fragment length polymorphism of the growth hormone gene in inbred rats

A novel restriction fragment length polymorphism in inbred rats was detected by Southern blot analysis with rat growth hormone cDNA as a probe. Four alleles, characterized by PstI fragments of 1.2, 1.1, 0.9, and 0.7 kb, respectively, were detected in 27 strains examined. The same distribution of polymorphisms was observed on digestion of DNAs of these strains with three other enzymes, PvuII, HindIII, and BamHI. Moreover, the same differences in length of allelic restriction fragments were obtained with these restriction enzymes as with PstI. These findings suggested that the polymorphism was caused by insertion or deletion of variable DNA segments in the second intron of the growth hormone gene. Linkage analyses using backcross progeny provided no evidence for close linkage between the restriction fragment length polymorphism locus and 10 other loci examined.     

Characterization of the cDNA and Gene Encoding Human PDE3B, the cGIP1 Isoform of the Human Cyclic GMP-Inhibited Cyclic Nucleotide Phosphodiesterase Family

Two distinct PDE3 [cyclic GMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE)] isoforms, cGIP1 and cGIP2, have been identified. Here we report cloning of the cDNA and gene encoding human (H)cGIP1 (classified as PDE3B). The cDNA encodes a protein of 1112 amino acids (123 kDa). Northern blots indicate that its mRNA is expressed in several adipose tissue depots. The human PDE3B gene is composed of 16 exons spanning more than 114 kb and was localized to chromosome 11p15 byin situhybridization. Exon/intron boundaries were determined, and genetic polymorphism, confirmed by single-strand conformational polymorphism of DNA from 25 healthy subjects, was demonstrated in exon 4 at nucleotide 1389 (A/G). Two polymorphic dinucleotide repeat sequences were identified in introns 5 and 12.     

Mucin 4 (MUC4) gene: regional assignment (3q29) and RFLP analysis.

The use of a probe (JER64) containing a mucin 4 (MUC4) cDNA insert of 1.83 kb allowed to assign by in situ hybridization, the MUC4 gene to 3q29. This probe detected RFLPs with all restriction enzymes used (BamHI, HindIII, PstI, EcoRI, and TaqI). Particularly numerous alleles were observed with PstI, EcoRI and TaqI, in a small sample of unrelated DNAs (25 digested with PstI, 8 with EcoRI and 8 with TaqI). The PIC values were 0.69, 0.63 and 0.70 for PstI, EcoRI and TaqI respectively. The polymorphisms observed of variable number of tandem repeat (VNTR) type are in relation with the presence of tandemly repeated nucleotide sequences in MUC4 gene.     

Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.     

Complete nucleotide sequence of the high molecular weight human IGF-I mRNA

IGF-I gene expression in mammals typically results in multiple mRNA species ranging in size between 0.7 and 7.6 kb. The smaller mRNA species have largely been characterized by the analysis of nearly full-length cDNAs. This report describes the first complete sequence of the prominent high molecular weight (7.6 kb) IGF-I mRNA species. Isolation and nucleotide sequence analysis of cDNA clones from human adult liver and uterus leiomyoma cDNA libraries resulted in a 7236 bp long sequence followed by a poly(A) tail. The sequence data, in combination with structural analysis of the human IGF-I gene, show that the 7.6 kb human IGF-I mRNA contains 6611 bp of untranslated 3' terminal sequence derived from a single exon. Alternate employment of two polyadenylation signals within the sequence transcribed from this exon generates two mRNAs of 1.1 and 7.6 kb.     

Characterization, expression and localization of S-adenosylhomocysteine hydrolase from amphioxus Branchiostoma belcheri tsingtaunese

A cDNA clone encoding AmphiSAHH [amphioxus SAHH (S-adenosylhomocysteine hydrolase)] protein was isolated from a cDNA library from the gut of Branchiostoma belcheri tsingtaunese. It contained a 1305 bp open reading frame corresponding to a deduced protein of 434 amino acid residues, with a predicted molecular mass of approx. 47.8 kDa. Phylogenetic analysis showed that AmphiSAHH and sea-urchin SAHH joined together and positioned at the base of the vertebrate SAHH clade, suggesting that both AmphiSAHH and sea-urchin SAHH might share some characteristics of the archetype of vertebrate SAHH proteins. The genomic DNA sequence of AmphiSAHH contained eight exons and seven introns, which was similar to B. floridae and sea-urchin SAHH exon/intron organization. Sequence comparison suggested the evolutionary appearance of the ten exon/nine intron organization of SAHH genes after the split of invertebrates and vertebrates, after which it has been highly conserved. AmphiSAHH has been successfully expressed in Escherichia coli and purified. Western blotting confirmed that the enzyme has a native molecular mass of approx. 48 kDa, and the catalytic activities and NAD(+)/NADH binding affinity of recombinant AmphiSAHH were measured. Immunohistochemistry analysis showed that SAHH was strongly expressed in hepatic caecum, gill, spermary and ovary of amphioxus.     

Loss ofHindIII cleavage sites in thed-amino acid oxidase gene in some inbred strains of mice

Summary d-Amino acid oxidase cDNA was amplified by a polymerase chain reaction using RNA extracted from the mouse kidney. When digested withHindIII, the cDNAs of the BALB/c and ddY/DAO^– mice were cleaved into two fragments whereas the cDNA of the ddY/DAO⁺ mice was not. Sequencing revealed that nucleotide-471 of the cDNAs was G in the BALB/c and ddY/DAO^– mice whereas it was substituted for C in the ddY/DAO⁺ mice. This base substitution was the cause of the failure of the cleavage of the cDNA of the ddY/DAO⁺ mice.Examination of other strains of inbred mice showed thatd-amino-acid oxidase cDNAs of A/J, AKR, C57BL/6, CD-1, CF#1, ICR, DBA/2, NZB and NZW mice were cleaved withHindIII into two fragments whereas those of C3H/He, CBA/J and NC mice were not. Genomic DNAs extracted from the mice of these 15 strains were digested withHindIII and hybridized withd-amino-acid oxidase cDNA. A 18.2-kb fragment hybridized with the probe in the C3H/He, CBA/J, ddY/DAO⁺ and NC mice whereas two fragments of 12 kb and 6.2 kb hybridized in the other mice. These results are consistent with those of the cDNAs, confirming the loss of theHindIII cleavage site in the C3H/He, CBA/J, ddY/DAO⁺ and NC mice. The Southern hybridization revealed a loss of a differentHindIII cleavage site in the A/J, AKR, C57BL/6, DBA/2, ICR and NZB mice.The substitution at nucleotide-471 should cause a substitution of an amino acid residue. However, this substitution did not affect catalytic activity ofd-amino acid oxidase.     

Molecular cloning and characterization of the complementary DNA and gene coding for the B-chain of subcomponent C1q of the human complement system.

Plasmid clones containing cDNA coding for the B-chain of human Clq were isolated from a liver cDNA library. The longest cDNA insert isolated contained all the coding sequence for amino acid residues B1 to B226 plus a 3' non-translated region of 264 nucleotides that extended into the poly(A) tail, thus accounting for 950 nucleotides of the mRNA. The B-chain mRNA was estimated by Northern-blot analysis to be 1.46 kb (kilobases) long, which indicated that approx. 500 bases were not accounted for in the cDNA clone. A cosmid clone containing the C1q-B chain gene was isolated from a human genomic DNA library. The precise 5' limit of gene was not established, but from the data available it appears that the gene is approx. 2.6 kb long. The coding sequence for residues B1 to B226 in the gene is interrupted by one intron, of 1.1 kb, which is located within the codon coding for glycine at position B36. This glycine residue is located in the middle of the triple-helical regions found in C1q at exactly the position where there is an unusual structural feature, i.e. a bend in each of the helical regions brought about by the interruption of the Gly-Xaa-Yaa repeating triplet sequences in the A- and C-chains and the presence of an 'extra' triplet in the B-chain. Nucleotide sequencing of the 5' end of the gene indicates the presence of a predominantly hydrophobic stretch of 29 amino acids, immediately before residue B1, which could serve as a signal peptide.     

Gene structure of human and mouse methylenetetrahydrofolate reductase (MTHFR)

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine. A human cDNA for MTHFR, 2.2 kb in length, has been expressed and shown to result in a catalytically active enzyme of approximately 70 kDa. Fifteen mutations have been identified in the MTHFR gene: 14 rare mutations associated with severe enzymatic deficiency and 1 common variant associated with a milder deficiency. The common polymorphism has been implicated in three multifactorial diseases: occlusive vascular disease, neural tube defects, and colon cancer. The human gene has been mapped to chromosomal region 1p36.3 while the mouse gene has been localized to distal Chromosome (Chr) 4. Here we report the isolation and characterization of the human and mouse genes for MTHFR. A human genomic clone (17 kb) was found to contain the entire cDNA sequence of 2.2 kb; there were 11 exons ranging in size from 102 bp to 432 bp. Intron sizes ranged from 250 bp to 1.5 kb with one exception of 4.2 kb. The mouse genomic clones (19 kb) start 7 kb 5′ exon 1 and extend to the end of the coding sequence. The mouse amino acid sequence is approximately 90% identical to the corresponding human sequence. The exon sizes, locations of intronic boundaries, and intron sizes are also quite similar between the two species. The availability of human genomic clones has been useful in designing primers for exon amplification and mutation detection. The mouse genomic clones will be helpful in designing constructs for gene targeting and generation of mouse models for MTHFR deficiency. Received: 28 January 1998 / Accepted: 9 April 1998     

Genomic structure of the bovine PrP gene and complete nucleotide sequence of bovine PrP cDNA

The present authors previously reported the nucleotide sequence of the 5' half of a cDNA encoding bovine prion protein (PrP) and the genomic structure of the bovine PrP gene encoding the 5'-untranslated region. Here they report the extent of intron 2 of the bovine PrP gene and the nucleotide sequence of the 3' half of bovine PrP cDNA that had not been determined before. This newly sequenced 3' half of the bovine PrP cDNA consisted of 2149 bp. The entire 3'-untranslated region (3'-UTR) was found to be encoded by a single exon, exon 3. One nucleotide polymorphism was found in the 3'-UTR. The length of intron 2 was estimated to be about 14 kbp. The structure of bovine PrP gene can be defined by combining the present results and previous reports on the bovine PrP gene.     

Structure of the human ornithine transcarbamylase gene

Complementary and genomic DNA clones corresponding to the human ornithine transcarbamylase (OTC) [EC 2.1.3.3]mRNA have been isolated and analyzed. The OTC gene is about 73 kilobase pairs (kb) long and contains 10 exons interrupted by 9 introns of highly variable sizes. The smallest intron is 80 base pairs and the largest, 21.7 kb. The 5'- and 3'-flanking regions, entire exons and all the exon/intron boundaries were sequenced. The nucleotide and deduced amino acid sequences of isolated OTC cDNAs as well as the corresponding regions of the genomic DNA were compared with those of human OTC cDNA (Horwich, A.L., Fenton, W.A., Williams, K.R., Kalousek, F., Kraus, J.P., Doolittle, R.F., Koningsberg, W., & Rosenberg, L.E. (1984) Science 224, 1068-1074). We found 20 nucleotide substitutions among these sequences, of which 6 related to amino acid changes. The nature of these nucleotide substitutions is discussed.     

外源性人TIMP-1基因在转基因小鼠染色体上的整合及定位

为探讨外源基因人基质金属蛋白酶组织抑制物-1（human tissue inhibitor of metalloproteinase-1, hTIMP-1）基因在转基因小鼠家系染色体上的整合和精确定位,应用Southrn印迹检测外源基因在染色体上整合的位点及拷贝数.结果表明,外源基因是以单拷贝、单位点形式整合;应用荧光原位杂交（fluorescence in situ hybridization, FISH）技术检测F4～F20代转基因小鼠中外源基因的整合.结果证明,该家系转基因小鼠自F4代起是纯合子,外源基因整合在17号染色体E区;反向PCR法（Inverse PCR, IPCR）克隆出约3.8 kb外源基因整合位点处的侧翼序列.分析表明,外源基因整合在17号染色体E1.3区,ALK（anaplastic lymphoma kinase, ALK）基因第23个内含子区域.结果提示,获得的转基因小鼠为纯系,外源基因hTIMP-1已稳定整合在转基因小鼠染色体上,并能遗传给后代.     

Identification and expression of cosmids with an allelic variant of class I alcohol dehydrogenase in transgenic mice

The mouse Adh1 gene exhibits tissue-specific regulation, is developmentally regulated, and is androgen regulated in kidney and adrenal tissue. To study this complex regulation phenotype a transgenic mouse approach has been used to investigate regulatory regions of the gene necessary for proper tissue expression and hormonal control. Transgenic mice have been produced with an Adh1 minigene as a reporter behind either 2.5- or 10 kb of 5'-flanking sequence [1]. Complete androgen regulation in kidney requires a region between -2.5 and -10 kb. A sequence extending to -10 kb does not confer liver expression in this minigene construct. B6.S mice express an electrophoretically variant protein resulting from a known nucleotide substitution resulting in a restriction endonuclease length polymorphism. Transgenic mice harboring B6.S cosmids can be studied for expression analysis at both protein and mRNA levels, identification of transgenic founders and inheritance studies are greatly facilitated by a PCR-restriction endonuclease cleavage approach, the entire mouse gene is used as a reporter, and the formation of heterodimeric enzyme molecules can be used to infer expression of the transgene in the proper cell types within a given tissue. Expression of a B6.S cosmid containing the entire Adh1 gene and 6 kb of 5'- and 21 kb of 3'-flanking region occurs in transgenic mice in a copy number dependent manner in a number of tissues, but expression in liver does not occur. The ability to analyze expression at the protein and mRNA levels has been confirmed using this system. Future directions will involve the use of large BAC clones modified by RARE cleavage to identify the liver specific elements necessary for expression.