The gene for human acidic fibroblast growth factor encodes two upstream exons alternatively spliced to the first coding exon

We have isolated two cDNA clones encoding human acidic fibroblast growth factor (aFGF) which represent the utilization of alternative upstream exons in aFGF mRNA. Isolation and sequence analysis of genomic clones spanning the first coding exon and each of the upstream sequences confirms that the divergent 5' sequences are separate exons, spliced alternatively to the first coding exon 34 nucleotides upstream of the initiator AUG codon. Restriction mapping of the genomic clones provides a minimum size estimate of 45 kilobase pairs for the aFGF locus.     

Human pregnancy-specific beta 1 glycoprotein is encoded by multiple genes localized on two chromosomes.

A human genomic library was screened with a mixture of two cDNA probes, with one covering the 5' coding sequence and the other containing the 3'-end portion of human pregnancy-specific beta 1 glycoprotein (SP1). Seventeen clones were identified, all of which carried insert fragments capable of hybridizing with the cDNA probe. Insert size of these clones varied from 15.0 to 19.8 kb. Partial restriction maps were constructed, which demonstrated the presence of at least seven groups of unique SP1 genomic clones and suggested the possibility of multiple genes coding for SP1. The multigene nature of SP1 was confirmed by hybridization of the SP1 cDNA probe to multiple bands on Southern blots of human genomic DNA. Further analysis with chromosomal DNA dot blot demonstrated the presence of homologous sequences on the X chromosome and autosomal chromosome 6. Thus, human SP1 is apparently coded for by more than one gene residing on the X and 6 chromosomes.     

Molecular cloning and sequence analysis of cDNA for human transferrin

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3' non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome.     

Cloning and sequence analysis of a cDNA for a rat liver glutathione S-transferase Yb subunit.

We have isolated a Yb-subunit cDNA clone from a GSH S-transferase (GST) cDNA library made from rat liver polysomal poly(A) RNAs. Sequence analysis of one of these cDNA, pGTR200, revealed an open reading frame of 218 amino acids of Mr = 25,915. The deduced sequence is in agreement with the 19 NH2-terminal residues for GST-A. The sequence of pGTR200 differs from another Yb cDNA, pGTA/C44 by four nucleotides and two amino acids in the coding region, thus revealing sequence microheterogeneity. The cDNA insert in pGTR200 also contains 36 nucleotides in the 5' noncoding region and a complete 3' noncoding region. The Yb subunit cDNA shares very limited homology with those of the Ya or Yc cDNAs, but has relatively higher sequence homology to the placental subunit Yp clone pGP5. The mRNA of pGTR200 is not expressed abundantly in rat hearts and seminal vesicles. Therefore, the GST subunit sequence of pGTR200 probably represents a basic Yb subunit. Genomic DNA hybridization patterns showed a complexity consistent with having a multigene family for Yb subunits. Comparison of the amino acid sequences of the Ya, Yb, Yc, and Yp subunits revealed significant conservation of amino acids (approximately 29%) throughout the coding sequences. These results indicate that the rat GSTs are products of at least four different genes that may constitute a supergene family.     

Nucleotide sequence of the gene for human factor IX (antihemophilic factor B)

Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z-DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin.     

Multiple polyadenylation sites downstream from the human aFGF gene encoding acidic fibroblast growth factor

We previously reported the isolation of two partial cDNA clones encoding human acidic fibroblast growth factor (aFGF). The nucleotide (nt) sequence throughout the coding region and the deduced amino acid sequence were presented [Jaye et al., Science 233 (1986) 541-545]. In this report, the isolation of additional aFGF cDNA clones and their nt sequence are presented. The human aFGF gene is shown to encode at least four functional polyadenylation sites and multiple regulatory sequences within the 3'-untranslated region. The aFGF open reading frame resides approx. 3100 bp upstream from the most frequently utilized 3' processing and polyadenylation site. Several less abundant cDNA clones provide evidence of polyadenylation at three less distal sites, which are colinear with genomic DNA. Northern-blot analysis reveals three detectable mRNA species, whose sizes and intensities correlate with the length and relative abundance of cDNA clones representing them.     

Structure of immunoglobulin gamma 2b heavy chain gene cloned from mouse embryo gene library.

A mouse DNA clone containing the constant part of the immunoglobulin gamma 2b heavy chain was isolated from a mouse gene library. The library was constructed in Charon 4A from a partial EcoRI digest of mouse embryo DNA and was screened with a plasmid (p gamma (11)7) containing a cDNA insert of the heavy chain constant region of the plasmacytoma MPC-11 (1). The Charon 4A clone contains a 14 kb insert which is cleaved by EcoRI into a 6.8 kb and 7.2 kb fragments, of which only the 6.8 kb contains the sequence for gamma 2b heavy chain. Restriction analysis and partial sequence of the insert in p gamma (11) 7 enabled us to obtain three fragments corresponding to the 5' (amino acid 161-302) middle (amino acid 302-443) and 3' (mostly non coding 107 bp) regions of the constant region. Restriction analysis of the Charon 4A clone and hybridisation to these nick translated fragments revealed that the gamma 2b constant region gene contains about 1.5 kb and has three intervening sequences.     

Cloning and sequence analysis of a cDNA from human ovarian granulosa cells encoding the C-terminal part of human elongation factor 2

A cDNA clone, pHGR81, encoding 358 amino-acid residues of the C-terminal region of human elongation factor 2 (EF-2), was isolated from a human ovarian granulosa cell cDNA library. The deduced amino-acid sequence of pHGR81, when compared with the known identical amino-acid sequences of hamster as well as rat EF-2 revealed a substitution of a glutamine by an alanine residue in the partially determined human sequence. The 15 amino-acid-residue sequence comprising the histidine-715, supposed to be of importance for the biological function of EF-2, is preserved in human EF-2. The coding region of the cDNA insert of pHGR81 displays a homology of 87% to hamster and of 88% to rat EF-2 cDNA. In Northern-transfer analysis, pHGR81 specifically hybridizes with an mRNA species of 3.1 kb.     

Gene transfer, expression, and molecular cloning of the human transferrin receptor gene

We describe the molecular cloning of the human transferrin receptor gene by a gene transfer approach. Mouse Ltk- cells were cotransformed with the herpes simplex thymidine kinase gene and total human DNA. Transformants expressing human transferrin receptor were isolated by selection on hypoxanthine/aminopterin/thymidine (HAT) medium and fluorescence-activated cell sorting of HAT-resistant cells. Thirty-four kilobases of human DNA was isolated by screening a genomic library constructed from the DNA of a secondary transformant. Gene transfer of the cloned DNA established that 31 kb of DNA was sufficient to encode the receptor. A probe from the 5' end of the gene was used to isolate a cDNA clone with an insert of 4.9 kb. Hybridization of the cDNA to the cloned genomic DNA revealed a minimum of 12 exons. They extend over the entire 31 kb of expressing DNA and over 2 kb of adjacent 3' untranslated sequences that are not required for receptor expression in L cells.     

Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter

We present the complete sequence of a cDNA encoding the human amiloride-sensitive Na+/H+ antiporter. After functional complementation of a mouse fibroblast mutant by gene transfer, we isolated a 0.8 kb genomic probe from a third-cycle mouse transformant. The probe detects gene amplification in Na+/H+ antiporter "overexpressers" and a single class of mRNA of ca. 5.6 kb in human, mouse, and hamster cells. With this probe we isolated a 4 kb cDNA from a library constructed from a mouse transformant in which the transfected human gene was amplified. This cDNA includes a noncoding leader of 407 bp, a 2682 bp open reading frame, and a 3' noncoding sequence containing a mouse B1 repeated element. The amino acid sequence predicts a protein of Mr = 99,354 with an N-terminal amphipathic domain that contains 10 putative transmembrane-spanning segments and two potential glycosylation sites, followed by a hydrophilic stretch of 395 residues, presumably cytoplasmic. Stable expression of the transfected cDNA in Na+/H+ antiporter-deficient cells restored the key functional features of this transporter: H+i-activated Na+ influx, amiloride sensitivity, and pHi regulation.     

Characterization of a genomic and cDNA clone coding for the thioesterase domain and 3' noncoding region of the chicken liver fatty acid synthase gene

The fatty acid synthase (FAS) of animal tissue is a dimer of two identical subunits, each with a Mr of 260,000. The subunit is a single multifunctional protein having seven catalytic activities and a site for binding of the prosthetic group 4'-phosphopantetheine. The mRNA coding for the subunit has an estimated size of 10-16 kb, which is about twice the number of nucleotides needed to code for the estimated 2300 amino acids. We have isolated a positive clone, lambda CFAS, containing FAS gene sequences by screening a chicken genomic library with a segment of a 3' untranslated region of goose fatty acid synthase cDNA clone, pGFAS3, as a hybridization probe. The DNA insert in lambda CFAS hybridizes with synthetic oligonucleotide probes prepared according to the known amino acid sequence of the thioesterase component of the chicken liver fatty acid synthase [Yang, C.-Y., Huang, W.-Y., Chirala, S., & Wakil, S.J. (1988) Biochemistry (preceding paper in this issue)]. Further characterization of the DNA insert shows that the lambda CFAS clone contains about a 4.7-kbp segment from the 3' end of the chicken FAS gene that codes for a portion of the thioesterase domain. Complete sequence analyses of this segment including S1 nuclease mapping, showed that the lambda CFAS clone contains the entire 3' untranslated region of the chicken FAS gene and three exons that code for 162 amino acids of the thioesterase domain from the COOH-terminal end of the fatty acid synthase. Using the exon region of the genomic clone, we were able to isolate a cDNA clone that codes for the entire thioesterase domain of chicken liver fatty acid synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The nucleotide sequence of a full length cDNA clone encoding rat liver urate oxidase

Recently we reported the sequence of a cDNA clone (pUOX-1), isolated from a lambda gt11 cDNA library, which encoded for rat liver urate oxidase (EC 1.7.3.3), but this clone lacked the nucleotide sequences encoding the N-terminal region for this enzyme. Using the cDNA insert from the pUOX-1 clone as a probe, we have now isolated a full length cDNA clone, pUOX-2, from a lambda gt10 library by plaque hybridization. Nucleotide sequence analysis of the pUOX-2 clone showed that it has 1379 base pairs with an open reading frame coding for 303 amino acid residues corresponding to a molecular mass of 34,931 daltons. In addition to the open reading frame the pUOX-2 contains 439 bp of 3'-untranslated and 41 bp of 5'-untranslated sequences. The consensus polyadenylation signal AATAAA precedes a stretch of poly(A)+ residues at the 3' end.     

Isolation and characteristics of clones complementary to mRNA of the murine cellular tumor antigen p53

43 cDNA clones specific for murine cellular tumor antigen p53 were isolated from a library constructed using 17S fraction of mRNA from the SV40 transformed murine fibroblasts (SVT2). These clones contain the whole coding region of p53 mRNA and the most of non-translated sequences. A plasmid containing 1.8 kb insert of p53 cDNA was constructed. The p53 specific insert in this plasmid was colinear with p53 mRNA, as revealed by S1 nuclease analysis. 5'-region of the p53 gene comprising non-translated and promoter areas was cloned from the mouse genomic library. A combined clone containing promoter and the whole region, corresponding to p53 mRNA has been constructed.     

Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences.

We have constructed a chimeric thymidine kinase (TK) minigene, pHe delta 6Ha, which combines the complete coding and 3' noncoding regions of a Chinese hamster TK cDNA with the promoter region and 5' untranslated region of the TK gene of herpes simplex virus type 1. We have transformed rat 4 cells to Tk+ with this gene and analyzed the pattern of TK gene expression in these transformants under various conditions of in vitro cell culture. We find that TK gene expression in these Tk+ transformants is growth phase dependent, responsive to adenovirus 5 infection, and indistinguishable in character under a variety of cell culture conditions from the pattern of TK gene expression in rat 4 cells transformed to Tk+ with the genomic Chinese hamster TK gene clone lambda HaTK.5. We are led to the conclusion that the genetic elements which mediate growth phase-dependent TK gene expression are contained entirely within the sequences of the mature cytoplasmic hamster TK mRNA.