The DNA sequence of Bombyx mori fibroin gene including the 5' flanking, mRNA coding, entire intervening and fibroin protein coding regions.

Sequence analysis of the cloned genomic fibroin gene and cDNA containing the sequence complementary to fibroin mRNA has been carried out for the regions covering the 5′ flanking, mRNA coding, entire intervening sequence and its borders, and fibroin coding sequences. The sequences determined on the gene extend from nucleotide ?552 to +1497 (assigning +1 to the cap locus); sequence analysis of the cDNA has confirmed our previous mapping of the cap locus (Tsujimoto and Suzuki, 1979). Comparison of the nucleotide sequence of the genomic gene with that of cDNA has confirmed the existence of an intervening sequence 970 bases long. The sequence comparison also pinpointed the 5′ coding-intervening junction at +64?66 and the 3′ intervening-coding junction at +1034?1036. Both the 5′ and 3′ junctions of the fibroin gene (insect) share homologous segments of about 10 nucleotides each with the published sequences of β-globin (mammal), immunoglobulin (mammal) and ovalbumin (avian) genes. A long inverted repeat sequence (17 of 23 base match) has been found next to the junctions within the intervening sequence of the fibroin gene. The repetitious sequence that codes for the Gly-Ala peptide characteristic of fibroin protein begins at position +1448. The characteristics of the N terminal portion of fibroin protein (or its precursor) are discussed, as are the features of the 5′ flanking sequence of the gene and the mRNA sequence (with special attention to the putative promoter sequence for the gene), the possible secondary structure and a sequence complementary to the 3′ end of 18S ribosomal RNA at the 5′ proximal region of fibroin mRNA.     

Analysis of the regions flanking the human insulin gene and sequence of an Alu family member.

The regions around the human insulin gene have been studied by heteroduplex, hybridization and sequence analysis. These studies indicated that there is a region of heterogeneous length located approximately 700 bp before the 5' end of the gene; and that the 19 kb of cloned DNA which includes the 1430 bp insulin gene as well as 5650 bp before and 11,500 bp after the gene is single copy sequence except for 500 bp located 6000 bp from the 3' end of the gene. This 500 bp segment contains a member of the Alu family of dispersed middle repetitive sequences as well as another less highly repeated homopolymeric segment. The sequence of this region was determined. This Alu repeat is bordered by 19 bp direct repeats and also contains an 83 bp sequence which is present twice. The regions flanking the human and rat I insulin genes were compared by heteroduplex analysis to localize homologous sequences in the flanking regions which could be involved in the regulation of insulin biosynthesis. The homology between the two genes is restricted to the region encoding preproinsulin and a short region of approximately 60 bp flanking the 5' side of the genes.     

The human alpha-fetoprotein gene. Sequence organization and the 5' flanking region

The human alpha-fetoprotein (AFP) gene was isolated into three overlapping clones in bacteriophage lambda vectors and its sequence organization analyzed by restriction endonuclease mapping and nucleotide sequencing. The human AFP gene is about 20 kilobase pairs long and contains 15 exons and 14 introns. The overall organization of the human AFP gene is similar to that of the mouse AFP gene, with all but two exons showing identical sizes. Nucleotide sequences at all exon/intron junctions display similarity to the consensus boundary sequence (Breathnach, R., and Chambon, P. (1981) Annu. Rev. Biochem. 50, 349-383), with the GT-AG rule applied to the splicing point. The cap site maps 44 nucleotides upstream from the translation initiation site. The "TATA box" is located 27 nucleotides upstream from the putative cap site and is flanked by sequences with dyad symmetry. The TATA box can thus be placed in the loop portion of a possible stem-loop structure formed by intrastrand base-pairing. Other characteristic nucleotide sequences in the 5' flanking region include a CCAAC pentamer, a 14-base pair (bp) enhancer-like sequence, and a 9-bp sequence homologous to the glucocorticoid responsive element. A long (90 bp) direct repeat and several alternating purine/pyrimidine sequences are also present in the 5' flanking region. A 736-bp sequence of the 5' flanking region adjacent to the cap site of the human AFP gene shows a 61% similarity with the corresponding region of the mouse AFP gene. There are two Alu family sequences and two poly(dT-dG) repeats in the human AFP gene that show different distribution patterns from those in the mouse AFP gene.     

Isolation and characterization of genomic DNA coding for alpha 2 type I collagen.

We have isolated and characterized a segment of the chick alpha 2 collagen gene by screening a library of chick genomic fragments using as hybridization probe an alpha 2 collagen cDNA clone. Several clones were isolated and one of them, lambda gCOL 204, was used for further studies. The DNA of lambda gCOL 204 hybridizes to a unique species of mRNA the size of alpha 2 collagen mRNA. This mRNA can be translated into a unique polypeptide which comigrates in SDS-gel electrophoresis with pro-alpha 2 collagen. Electron microscopic analysis by R-loop technique indicates that lambda gCOL 204 contains 7Kb of the alpha 2 collagen gene. This 7 Kb piece constitutes the 3' end of the gene. The same clone also contains 9 Kb of DNA that is immediately adjacent to the 3' end of the alpha 2 collagen gene. The cloned segment of the alpha 2 collagen gene is interrupted by 8 intervening sequences of various lengths. The coding sequences for collagen in this clone add up to approximately 1,800 bp, which correspond to about 1/3 of alpha 2 collagen mRNA. DNA sequence analysis of a small coding segment of lambda g COL 204 reveals a characteristic collagen type sequence which encodes for an amino acid sequence identical to a sequence found in calf alpha 2 collagen. The sequence of this region of the protein has not yet been determined for the chick alpha 2 collagen.     

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

Isolation and characterisation of genomic and cDNA clones for an androgen-regulated secretory protein of rat seminal vesicles.

Testosterone controls the synthesis of seminal vesicle protein F in male rats by regulating the cellular concentration of its mRNA (mRNAF). Phage lambda recombinants have been isolated containing the complete F gene. In addition plasmids have been constructed containing cDNAF sequences some of which are probably full-length (approximately 700 bp). Detailed restriction mapping shows that the F gene is 1.7 kbp long and contains approximately 1.0 kbp of intervening sequence arranged in at least two introns (420 bp and 600 bp). Part of cDNAF has been sequenced showing that the terminal 125 bp of the 3' untranslated region of mRNAF has substantial (greater than 70%) sequence homology with the 3' end of the mRNA coding for another androgen-dependent seminal vesicle protein (protein S). The cloned F gene has been detected in liver and seminal vesicle DNA along with an homologous but structurally different gene. The hormonal control of mRNAF was examined with cDNAF. A pronounced (approximately 3000-fold) differential response to testosterone was observed.     

The histidine tRNA genes of yeast

Yeast has at least seven nuclear histidine tRNA genes although there is a single tRNAHis. We have sequenced three of the histidine tRNA genes. The genes have identical coding sequences and the DNA anti-codon sequence GTG corresponds to the GUG anti-codon in tRNAHis. None of the three yeast histidine tRNA genes has an intervening sequence. Two of the three genes contain repeated DNA elements in the region adjacent to the 5' end of the histidine tRNA gene. One of the elements, sigma, is 18 base pairs (bp) from the 5' end of each of these genes, sigma elements are highly conserved and flanked by 5-bp repeats. The other element, delta, is at variable distances from the tRNA gene; one is 439 bp from a histidine tRNA gene and the other is 52 bp from a histidine tRNA gene. These solo delta elements are quite divergent when compared with delta s associated with transposon yeast elements and are not flanked by 5-bp repeats.     

Tandem repeats within the inverted terminal repetition of vaccinia virus DNA

A tandemly repeated sequence within the genome of vaccinia virus is cut to fragments of approximately 70 bp by Hinf I, Taq I or Mbo II. The 70 bp repetition was localized within the much larger (10,300 bp) inverted terminal repetition by restriction analysis of cloned DNA fragments and by hybridization of the purified 70 bp repeat to vaccinia virus DNA restriction fragments. The molar abundance of the 70 bp fragment corresponds to a 30 fold repetition at each end of the genome. The repeating restriction endonuclease sites were mapped by agarose gel electrophoresis of partial Hinf I digests of the terminally labeled cloned DNA fragment. The first of 13 repetitive Hinf I sites occurred approximately 150 bp from the end of the cloned DNA. After an intervening sequence of approximately 435 bp, a second series of 17 repetitive Hinf I sites occurred. The DNA between the two blocks of repetitions has a unique sequence containing single Dde I, Alu I and Sau 3A sites. Tandem repeats within the inverted terminal repetition could serve to accelerate self-annealing of single strands of DNA to form circular structures during replication.     

Nucleotide sequence of the gene for human prothrombin

A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were then compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases.     

Cloning and analysis of the pseudogene for human epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT)

1. This gene completely lacks the intervening sequences. 2. This gene is truncated at the 5' end peptide encoding region by 433 base pairs (bp). 3. The 502 bp of this gene containing poly(A) signal are completely identical to the 3' half of mRNA encoding region of functional gene. 4. This gene has a poly(A) tail and is flanked by direct repeat of 6 bp. 5. Here we report for the first time the complete sequence of a human pseudogene for phenylethanolamine N-methyltransferase and this is the first report of cloning of pseudogene for catecholamine biosynthetic enzymes.     

The Bos taurus casein genes. Isolation and characterization of the kappa-casein gene

A region spanning 25 kb of genomic DNA containing the kappa-casein gene, has been isolated from two genomic libraries in EMBL3 and EMBL4 phage vectors. Five phage clones containing kappa-casein gene have been found. Gene organisation has been determined using restriction mapping and a partial sequencing the 5' and 3' flanking regions. The kappa-casein gene includes 5 exons, the first of them coding for 64 nucleotides from the 5' untranslated mRNA zone. The gene is 12.5 kb long, which is almost 16 times longer than the corresponding mRNA. The first intron spans 2.5 kb, the second is the largest one and spans 5.5 kb. The 5' flanking region sequence has been analysed; it contains a TATA box from -30 to -25 bp, somewhat different from the canonic sequence, and a CAAT box at -80 bp.