Showing papers on "Exon published in 1986"

Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma.

Abstract: We have determined that the bcl-2 (B-cell leukemia/lymphoma 2) gene is transcribed into three overlapping mRNAs, and we have cloned bcl-2 cDNA sequences. Sequence analysis of the bcl-2 cDNA clones and comparison of their sequences to their genomic counterparts indicate that the bcl-2 gene contains at least two exons. The three bcl-2 transcripts, which are 8.5, 5.5, and 3.5 kilobases (kb) long, overlap within the first exon, but only the 8.5-kb and 5.5-kb transcripts contain sequences of the second exon. The 8.5-kb and 5.5-kb transcripts seem to use different polyadenylylation sites. Sequence analysis of the cDNA clones corresponding to the 5.5-kb and 3.5-kb mRNAs indicates that the two bcl-2 transcripts carry two overlapping open reading frames, one of which is 717 nucleotides long and codes for a protein (bcl-2 alpha) of 239 amino acids and a molecular mass of 26 kDa, while the other codes for a protein of 205 amino acids (bcl-2 beta, molecular mass 22 kDa) that is identical to bcl-2 alpha except at the carboxyl terminus. The bcl-2 protein products in follicular lymphomas with or without bcl-2 rearrangements are identical to the normal bcl-2 products.

Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor.

Abstract: Basic and acidic fibroblast growth factors (FGF's) are potent mitogens for capillary endothelial cells in vitro, stimulate angiogenesis in vivo, and may participate in tissue repair. An oligonucleotide probe for bovine basic FGF was designed from the nucleotide sequence of the amino-terminal exon of bovine acidic FGF, taking into account the 55 percent amino acid sequence homology between the two factors. With this oligonucleotide probe, a full length complementary DNA for basic FGF was isolated from bovine pituitary. Basic FGF in bovine hypothalamus was shown to be encoded by a single 5.0-kilobase messenger RNA; in a human hepatoma cell line, both 4.6- and 2.2-kilobase basic FGF messenger RNA's were present. Both growth factors seem to be synthesized with short amino-terminal extensions that are not found on the isolated forms for which the amino acid sequences have been determined. Neither basic nor acidic FGF has a classic signal peptide.

A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells.

Abstract: The c-myc gene product is a nuclear protein1,2 expressed in a wide variety of cell types3. It has been implicated in the control of normal cell growth as well as transformation4–7, but its exact function is unknown. When the human promyelocytic leukaemia cell line HL60 is treated with retinoic acid, the cells differentiate into granulocytes, and there is a reduction in steady state c-myc RNA of more than 10-fold8. Nuclear runoff assays show that this reduction is caused by a corresponding decrease in the transcription of exon 2. However, only a minor decrease in exon 1 transcription is observed upon differentiation. In undifferentiated HL60 cells there is an approximately 3-fold molar excess of exon 1 transcription over exon 2, and this excess increases to about 15-fold in differentiated cells. This observation suggests that a major component of c-myc transcriptional down-regulation in HL60 cells is at the level of elongation rather than at the level of initiation. The position of the elongation block was mapped to the region of the boundary between exon 1 and intron 1. During HL60 differentiation, a DNase I hypersensitive site in the chromatin about 300 bases downstream of the 5′ end of of intron 1 increases in intensity relative to other sites, possibly reflecting events associated with the termination of transcription. Our runoff analysis also revealed transcription of both strands immediately upstream of exon 1 in HL60 cells. The sense strand transcription of this region produces a novel c-myc RNA which initiates several hundred bases upstream of the previously defined promoters and is found in a variety of cell types.

Regulation of insulin-like growth factor I gene expression by growth hormone.

Abstract: Recombinant clones containing exon 3 of the insulin-like growth factor I (IGF-I) gene were isolated from a mouse genomic library. These sequences were used to generate an RNA probe, which was used in a solution hybridization assay to quantitate IGF-I mRNA in various murine tissues as a function of growth hormone status. The liver is the major site of IGF-I synthesis and the level of IGF-I mRNA is regulated about 10-fold by growth hormone in the growth hormone-deficient lit/lit mouse. Nuclear run-on assays were used to show that growth hormone regulation is manifested in part at the transcriptional level. Growth hormone also affects the size distribution of hepatic IGF-I mRNAs. Pancreas showed the highest non-hepatic expression, but every tissue analyzed contained some IGF-I mRNA. Expression was not growth hormone-dependent in most non-hepatic tissues.

Complete nucleotide sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosome: a pseudogene and a genuine gene

Abstract: Two 21-hydroxylase [P-450(C21)] genes have been isolated from a human genomic library using a bovine P-450(C21) cDNA. The insert DNAs containing the P-450(C21) genes were also hybridized with the sequences of the 5' or 3' end regions of human C4 cDNA, indicating a close linkage of the P-450(C21) gene to the C4 gene. Sequence analysis has revealed that the two P-450(C21) genes are both approximately equal to 3.4 kilobases long and split into 10 exons. Comparing the two sequences, we found that the two genes are highly homologous including their introns and flanking sequences, but that three mutations render one of the two P-450(C21) genes nonfunctional--1 base insertion, an 8-base deletion, and a transition mutation--all of which may cause premature termination of the translation. Tandem arrangement of the highly homologous pseudo- and genuine genes in close proximity could account for the high incidence of P-450(C21) gene deficiency by homologous gene recombination.

Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

Abstract: We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.

Different Human Cervical Carcinoma Cell Lines Show Similar Transcription Patterns of Human Papillomavirus Type 18 Early Genes

Abstract: Transcription of human papillomavirus type 18 (HPV18) DNA in the human cervical carcinoma cell lines HeLa, C4-1 and SW756 was studied by nucleotide sequence analysis of HPV18-positive cDNA clones isolated from a HeLa, C4-1 and SW756 cDNA library, respectively, and the cDNA sequences were used to predict the potential encoded proteins. The cDNA clones from all three cell lines were found to be derived from virus-cell fusion transcripts in which 3'-terminal host cell sequences (different for each cell line) were spliced to 5'-terminal exon sequences from the HPV18 E6-E7-E1 region. Three different types of cDNA clones can be distinguished according to the splicing patterns observed in the 5' terminal HPV18 sequences. They carry as potential protein-coding regions the HPV18 specific open reading frames E6 and E6* (generated by splicing and identical with E6 up to the E6* splice junction), E7 and E1 (only in HeLa). Translation of specific cellular genes from the chimeric viral-cellular transcripts seems to be unlikely. The mapping of the 5'-ends of the virus-cell fusion transcripts indicates that transcription is initiated at a viral promoter. The similar patterns of HPV18 transcription in the three different cervical carcinoma cell lines suggest a functional role of HPV18 early genes for the malignant phenotype of these cells.

Isolation of the human gene for bone gla protein utilizing mouse and rat cDNA clones.

Abstract: cDNAs which encode bone gla protein (BGP), an abundant gamma-carboxylated protein of bone, have been cloned from rat and mouse osteosarcoma cell lines. DNA sequence analysis indicates that the cDNAs code for both the 50 (rat) or 46 (mouse) amino acids of the mature proteins and a 49 amino acid leader peptide. The leader peptide of each BGP includes the expected hydrophobic signal sequence and an apparent pro sequence. Although there is no homology between the mature forms of BGP and the gamma-carboxylated clotting factors, we note that there is some homology between their leader peptides. These cDNAs have been used to examine the modulation of BGP mRNA levels by osteoblastic cells in response to hormones. The cDNAs have also allowed isolation of the human BGP gene; analysis of this gene indicates the presence of four exons. Comparison of the exon structure of the BGP gene and the Factor IX (a gamma-carboxylated clotting factor) gene suggests that the exons encoding the part of the leader peptides presumably directing gamma-carboxylation arose from a common ancestral sequence.

Characterization of the human p53 gene.

Abstract: Cosmid and lambda clones containing the human p53 gene were isolated and characterized in detail. The gene is 20 kilobases (kb) long and has 11 exons, the first and second exons being separated by an intron of 10 kb. Restriction fragments upstream of sequences known to be within the first identified exon were tested for promoter activity by cloning them in front of the chloramphenicol acetyltransferase gene and transfecting the resulting constructs into HeLa cells. A 0.35-kb DNA fragment was identified that had promoter activity. Results of primer extension experiments indicated that the mRNA cap site falls within this fragment, as expected. Analysis of the sequence upstream of the presumptive cap site indicated that the human p53 promoter may be of an unusual type.

Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization

Abstract: Complementary DNA clones encoding the human kidney epidermal growth factor (EGF) precursor have been isolated and sequenced. They predict the sequence of a 1,207 amino acid protein which contains EGF flanked by polypeptide segments of 970 and 184 residues at its NH2- and COOH-termini, respectively. The structural organization of the human EGF precursor is similar to that previously described for the mouse protein and there is 66% identity between the two sequences. Transfection of COS-7 cells with the human EGF precursor cDNA linked to the SV40 early promoter indicate that it can be synthesized as a membrane protein with its NH2-terminus external to the cell surface. The human EGF precursor gene is approximately 110 kilobase pairs and has 24 exons. Its exon-intron organization revealed that various domains of the EGF precursor are encoded by individual exons. Moreover, 15 of the 24 exons encode protein segments that are homologous to sequences in other proteins. Exon duplication and shuffling appear to have played an important role in determining the present structure of this protein.

Isolation of the oncogene and epidermal growth factor-induced transin gene: complex control in rat fibroblasts

Abstract: Various oncogenes or epidermal growth factor (EGF) induce transcription of a 1.9-kilobase RNA (transin RNA) in rat fibroblasts. The induction by EGF can be blocked by cycloheximide. Thus the response of the transin gene to EGF appears to require de novo protein synthesis. Transin RNA induction is specific to EGF, as neither insulin, platelet-derived growth factor, fibroblast growth factor, nor transforming growth factor beta could elicit the same response. However, transforming growth factor beta could block the EGF induction of transin RNA. Whereas the calcium ionophore A23187 and the tumor promoter TPA, either alone or administered together, did not increase transin RNA levels, TPA could synergise with a serum factor to effect such an increase. Dibutyryl cyclic AMP also induced transin RNA. Treatment of cells with the microfilament-disrupting agent cytochalasin B, but not the microtubule-disrupting agent colcemid, resulted in an increase in transin RNA levels, suggesting a role for the cytoskeleton in control of transin gene expression. The transin RNA does not contain repeated sequences and appears to be encoded by a single-copy gene. The protein sequence encoded by the last four exons of the transin gene shows some homology to two regions of the heme-binding protein hemopexin.

Molecular analysis of the waxy locus of Zea mays

Abstract: The structure of the wild-type waxy (wx+) locus was determined by sequence analysis of both a genomic and an almost full-size cDNA clone. The coding region comprises 3,718 bp and is composed of 14 exons and 13 small introns. The exons and the promoter region are G/C rich (60%–80%). All three waxy transcripts analysed so far reveal different polyadenylation sites and corresponding polyadenylation signals. The smallest of these mRNAs has a size of 2,263 nucleotides. Northern blot analysis suggests that the tissue-specific expression of the locus is due to transcriptional control. The insertion sites of all transposable element induced waxy mutations analysed have been mapped precisely within the locus. N-terminal sequencing of the mature wx+ protein leads to the identification of a maize amyloplast-specific transit peptide of 72 aminoacid residues.

Human small-cell lung cancers show amplification and expression of the N-myc gene

Abstract: We have found that 6 of 31 independently derived human small-cell lung cancer (SCLC) cell lines have 5- to 170-fold amplified N-myc gene sequences. The amplification is seen with probes from two separate exons of N-myc, which are homologous to either the second or the third exon of the c-myc gene. Amplified N-myc sequences were found in a tumor cell line started prior to chemotherapy, in SCLC tumor samples harvested directly from tumor metastases at autopsy, and from a resected primary lung cancer. Several N-myc-amplified tumor cell lines also exhibited N-myc hybridizing fragments not in the germ-line position. In one patient's tumor, an additional amplified N-myc DNA fragment was observed and this fragment was heterogenously distributed in liver metastases. In contrast to SCLC with neuroendocrine properties, no non-small-cell lung cancer lines examined were found to have N-myc amplification. Fragments encoding two N-myc exons also detect increased amounts of a 3.1-kilobase N-myc mRNA in N-myc-amplified SCLC lines and in one cell line that does not show N-myc gene amplification. Both DNA and RNA hybridization experiments show that in any one SCLC cell line, only one myc-related gene is amplified and expressed. We conclude that N-myc amplification is both common and potentially significant in the tumorigenesis or tumor progression of SCLC.

Structure and gene organization of bovine neuromedin K precursor.

Abstract: cDNA and genomic DNA clones for the precursor of a mammalian neuropeptide tachykinin, neuromedin K, have been isolated and characterized by molecular cloning and sequence analysis. The deduced amino acid sequence indicates that the bovine neuromedin K precursor (preprotachykinin B) consists of 126 amino acid residues including a putative signal peptide. There are two preprotachykinin B mRNAs that differ only at the 5' extremity of the untranslated regions. The major mRNA species is encoded by seven exon sequences, while the minor species includes two extra 5' exon sequences and lacks the 5' terminus of the first exon sequence for the major mRNA species. The above gene organization for the major preprotachykinin B mRNA closely resembles that for the preprotachykinin A mRNA encoding the precursor for substance P and substance K. This structural resemblance strongly suggests that the two preprotachykinin genes have evolved from a common ancestor gene. Furthermore, we have found that the preprotachykinin A and B mRNAs markedly differ in their major expression sites. The results described thus indicate that the diversity of the mammalian tachykinin system has been acquired through various cellular mechanisms including gene duplication and differential expression of duplicated genes.