Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences.

Two functional cytosolic thymidine kinase (tk) cDNA clones were isolated from a mouse L-cell library. An RNA blot analysis indicated that one of these clones contains a nearly full-length tk sequence and that LTK- cells contain little or no TK message. The nucleotide sequences of both clones were determined, and the functional mouse tk cDNA contains 1,156 base pairs. An analysis of the sequence implied that there is an untranslated 32-nucleotide region at the 5' end of the mRNA, followed by an open reading frame of 699 nucleotides. The 3' untranslated region is 422 nucleotides long. Thus, the gene codes for a protein containing 233 amino acids, with a molecular weight of 25,873. A comparison of the coding sequences of the mouse tk cDNA with the human and chicken tk genes revealed about 86 and 70% homology, respectively. We also isolated the tk gene from a mouse C57BL/10J cosmid library. The structural organization was determined by restriction mapping, Southern blotting, and heteroduplex analysis of the cloned sequences, in combination with a mouse tk cDNA. The tk gene spans approximately 11 kilobases and contains at least five introns. Southern blot analysis revealed that this gene is deleted in mouse LTK- cells, consistent with the inability of these cells to synthesize TK message. This analysis also showed that tk-related sequences are present in the genomes of several mouse strains, as well as in LTK- cells. These segments may represent pseudogenes.     

Nucleotide sequence of cDNA clones of the murine myb proto-oncogene.

We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5' and 3' to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c-myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb.     

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.     

The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene

This paper presents the nucleotide sequence of the Herpes Simplex Virus thymidine kinase (tk) gene. The position on the DNA sequence corresponding to the 5' and 3' termini of tk messenger RNA have been mapped. The mRNA termini are separated by slightly more than 1,300 nucleotides. The same 2,300 nucleotide segment of tk coding strand DNA is fully protected from S1 nuclease digestion when hybridized to tk mRNA. The location and size of the mRNA-coding segment corresponds to a region of the viral DNA that is essential for tk gene expression in microinjected frog oocytes. The nucleotide sequence of the HSV tk gene exhibits an open translational reading frame of 376 codons that extends from the methionine codon most proximal to the 5' terminus of tk mRNA to a UGA stop codon approximately 70 nucleotides from the poly-A addition site. The results of these experiments indicate that the tk gene is not interrupted by intervening DNA sequences, and that certain oligonucleotide sequences adjacent to the termini of the tk gene are homologous to similarly positioned sequences common to structural genes of eukaryotic cells.     

The chicken carbonic anhydrase II gene: evidence for a recent shift in intron position.

The complete nucleotide sequence of the coding region of the chicken carbonic anhydrase II (CA II) gene has been determined from clones isolated from a chicken genomic library. The sequence of a nearly full length chicken CA II cDNA clone has also been obtained. The gene is approximately 17 kilobase pairs (kb) in size and codes for a protein that is comprised of 259 amino acid residues. The 5' flanking region contains consensus sequences commonly associated with eucaryotic genes transcribed by RNA polymerase II. Six introns ranging in size from 0.3 to 10.2 kb interrupt the gene. The number of introns as well as five of the six intron locations are conserved between the chicken and mouse CA II genes. The site of the fourth intron is shifted by 14 base pairs further 3' in the chicken and thus falls between codons 147 and 148 rather than within codon 143 as in the mouse gene. Measurements of CA II RNA levels in various cell types suggest that CA II RNA increases in parallel with globin RNA during erythropoiesis and exists only at low levels, if at all, in non-erythroid cells.     

Molecular cloning, sequence analysis, and functional expression of a novel growth regulator, oncostatin M.

Oncostatin M is a polypeptide of Mr approximately 28,000 that acts as a growth regulator for many cultured mammalian cells. We report the cDNA and genomic cloning, sequence analysis, and functional expression in heterologous cells of oncostatin M. cDNA clones were isolated from mRNA of U937 cells that had been induced to differentiate into macrophagelike cells by treatment with phorbol 12-myristate 13-acetate, and a genomic clone was also isolated from human brain DNA. Sequence analysis of these clones established the 1,814-base-pair cDNA sequence as well as exon boundaries. This sequence predicted that oncostatin M is synthesized as a 252-amino-acid polypeptide, with a 25-residue hydrophobic sequence resembling a signal peptide at the N terminus. The predicted oncostatin M amino acid sequence shared no homology with other known proteins, but the sequence of the 3' noncoding region of the cDNA contained an A + T-rich stretch with sequence motifs found in the 3' untranslated regions of many cytokine and lymphokine cDNAs. Oncostatin M mRNA of approximately 2 kilobase pairs was detected in phorbol 12-myristate 13-acetate-treated U937 cells and in activated human T cells. Transfection of cDNA encoding the oncostatin M precursor into COS cells resulted in the secretion of proteins with the structural and functional properties of oncostatin M. The unique amino acid sequence, expression by lymphoid cells, and growth-regulatory activities of oncostatin M suggest that it is a novel cytokine.     

Two genes encoding ''minor'' legumin polypeptides in pea (Pisum sativum L.). Characterization and complete sequence of the LegJ gene.

A genomic clone from pea (Pisum sativum L.) contains all of one gene encoding a 'minor' (B-type) legumin polypeptide, and most of a second very similar gene. The two genes, designated LegJ and LegK, are arranged in tandem, separated by approx. 6 kb. A complete sequence of gene LegJ and its flanking sequences is given, with as much of the sequence of gene LegK as is present on the genomic clone. Hybridization of 3' flanking sequence probes to seed mRNA, and sequence comparisons with cDNA species, suggested that gene LegJ, and probably gene LegK, was expressed. The partial amino acid sequences of 'minor' legumin alpha- and beta-polypeptides were used to confirm the identity of these genes. The transciption start in gene LegJ was mapped. The 5' flanking sequence of gene LegJ contains a sequence conserved in legumin genes from pea and other species, which is likely to have functional significance in control of gene expression. Sequence comparisons with legumin genes and cDNA species from Vicia faba and soya bean show that separation of legumin genes into A- and B-type subfamilies occurred before separation of the Viciae and Glycinae tribes.     

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)     

马铃薯卷叶病毒中国株（PLRV-Ch）ORF2a基因特征分析

马铃薯卷叶病毒 (ＰＬＲＶ)是正链ＲＮＡ病毒 ,属黄化病毒组[1 ] 严格虫传 ,分布广泛 ,难以控制 ,侵染马铃薯 ,给生产造成巨大损失。ＰＬＲＶ基因全长 6 0ｋｂ ,有 6个读码框架 ,其中ＯＲＦ2ａ是第二读框 ,全长 192 0ｂｐ ,编码一个 70ｋＤ的多肽。另外 ,ＯＲＦ2ａ在与ＯＲＦ2ｂ重叠处可发生移码继续转译 ,直到ＯＲＦ2ｂ的尾 ,转译产物为一条 118ｋＤ的多肽 ,该蛋白的Ｃ端与复制酶的序列具很大的同源性[2～ 4] 。Ｐｒｕｆｅｒ[5] 等和Ｋｕｊａｗａ[6] 等分别研究了ＰＬＲＶ基因组上ＯＲＦ2ａ与ＯＲＦ2ｂ重叠区附近与移码有关的滑动序列及其…     

Analysis of cDNAs of the proto-oncogene c-src: heterogeneity in 5'' exons and possible mechanism for the genesis of the 3'' end of v-src.

To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60c-src coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60c-src. Within the 3' noncoding region, a 39-bp sequence corresponding to the 3' end of the RSV v-src was detected 660 bases downstream of the pp60c-src termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5' region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5' exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. All of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon 1a, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain cDNA library. Exon UE2 is located at 8.5 kb upstream from exon 1. The precise locations of exons UEX and UEY have not been determined, but both are more than 12 kb upstream from exon 1. The existence and exon arrangements of these 5' cDNAs were further confirmed by RNase protection assays and polymerase chain reactions using specific primers. Our findings indicate that the heterogeneity in the 5' sequences of the c-src mRNAs results from differential splicing and perhaps use of distinct initiation sites. All of these RNAs have the potential of coding for pp60c-src, since their 5' exons are all eventually joined to exon 2.