摘要:

ABSTRACTWe have obtained a cDNA clone coding for human transforming growth factor (TGF)-β2. The clone was isolated from a tamoxifen-treated human prostatic adenocarcinoma cell line (PC-3) using oligonucleotide probes based on the partial amino acid sequence of purified TGF-β2. The cDNA sequence predicts that TGF-β2 is synthesized as a 442-amino-acid polypeptide precursor from which the mature 112-amino-acid TGF-β2 subunit is derived by proteolytic cleavage. The proteins coded for by the human TGF-β1 and TGF-β2 cDNAs show an overall homology of 41%. The mature and amino-terminal precursor regions show 71% and 31% homology, respectively. Northern blot analysis identified TGF-β2 transcripts of 4.1, 5.1, and 6.5 kb using mRNA from several different sources. Analysis of polyadenylated RNA from tamoxifen-treated PC-3 cells showed that these cells contain higher numbers of transcripts for TGF-β1 than for TGF-β2, although they produce more TGF-β2 protein than TGF-β1. This suggests that there is a post-transcriptional level of regulation for the production of thes

ISSN:1044-5498    

DOI:10.1089/dna.1988.7.1

年代:1988

数据来源: MAL

摘要:

ABSTRACTInsulin-like growth factor II (IGF-II) is regulated developmentally and hormonally in human fetal gonads and adrenals. The abundance of IGF-II mRNA is greatest in RNA from human fetal adrenals, followed by fetal liver, testis, placenta, and ovaries. Fetal testicular IGF-II mRNA decreases significantly with increasing gestational age, in parallel with our previous measurements of the mRNAs for the steroidogenic enzymes P450scc (cholesterol side-chain cleavage enzyme) and P450cl7 (17α-hydroxylase/17,20 lyase) (J. Clin. Endocrinol. Metab. 63, 1145, 1986). The abundances of P450scc and P450cl7 mRNAs in cultured fetal testis cells rose 2.5-fold (p<0.01) and 9.2-fold (p<0.001), respectively, in response to 0.5 mMcAMP, but the abundance of IGF-II mRNA was not affected. This suggests that the IGF-II gene is regulated differently in fetal testes than it is in fetal adrenals, placenta, or adult granulosa cells, where we have previously shown that ACTH, cAMP, and gonadotropins, respectively, increase IGF-II mRNA accumulation (Proc. Natl. Acad. Sci. USA 84, 1590, 1987). Exogenously added IGF-I and IGF-II had no effect on mRNAs for P450cl7 or P450c21 (21-hydroxylase), but decreased IGF-II mRNA in ACTH-stimulated fetal adrenal cells. Thus, the IGFs appear to exert short-loop feedback inhibition on accumulation of IGF-II mRNA

ISSN:1044-5498    

DOI:10.1089/dna.1988.7.9

年代:1988

数据来源: MAL

摘要:

ABSTRACTWe constructed a murine genomic DNA library in λ EMBL3 and have isolated and determined the nucleotide sequence of the murine thyrotropin β-subunit (TSHβ) gene. The cloned gene was derived from a thyrotropic tumor and had no detectable rearrangements when compared to the murine TSHβ gene in total genomic DNA. The murine TSHβ gene is 5 kb in size and consists of five exons and four introns. The 5′ untranslated region of the mRNA is encoded except for a single nucleotide by exons 1, 2, and 3. The protein-coding regions are encoded by exons 4 and 5 while the 3′ untranslated region is entirely contained in exon 5. Primer extension analysis using an exon 1-specific primer was used to map the 5′ end of the gene. Two transcriptional start sites are present in the murine TSHβ gene which appear to be positioned by two TATAAA sequences located 40 bp apart. In all, 99% of transcripts initiate at the downstream site. Transcription from both start sites is affected by thyroidal status in both murine pituitaries and in TtT97 thyrotropic tumors. Finally, sequences homologous with putative thyroid-responsive elements and cyclic AMP-responsive elements are present in the 5′-flanking region and may be important in regulating negative and positive effects on TSHβ g

ISSN:1044-5498    

DOI:10.1089/dna.1988.7.17

年代:1988

数据来源: MAL

摘要:

ABSTRACTThe amino acid sequence of human placental aromatase was determined in part (about 40%) by microsequencing methods. Using a region of overlapping peptide sequences, synthetic oligonucleotide probes were constructed and used to screen a human placental λgt-11 cDNA library. Of a number of positive clones, one containing a 2.4-kb insert was characterized further by restriction mapping and determination of its nucleotide sequence. The cDNA-deduced amino acid sequence is in perfect agreement with the peptide sequence data, confirming that the clone encodes for aromatase. The sequence contains a 3′ untranslated region of 1.2 kb, and an open-reading frame of 1.25 kb; approximately 0.3 kb is missing from the 5′ end of the coding region. While exhibiting no more than 20–30% sequence homology with other mammalian cytochromes P450, it contains the highly conserved heme-binding domain, thus confirming the essential structural requirements for this class of protein. Two cDNA fragments containing sequences coding for the amino- and carboxy-portions of the protein were used to probe for the human aromatase gene by Southern blotting. The results of these studies suggest the existence of at least two human aromatase genes. The gene encoding the aromatase cDNA we cloned was assigned to human chromosome 15 using somatic cell hybrids. This gene was mapped to band 15q21.1 byin situhybridization s

ISSN:1044-5498    

DOI:10.1089/dna.1988.7.27

年代:1988

数据来源: MAL

摘要:

ABSTRACTTwo cDNAs (pLM3c 4.1 and pLM3c 6.1) coding for rabbit cytochrome P450 3c were sequenced. cDNA 4.1 (1768 bp) exhibits an open reading frame from nucleotides 74 to 1576 encoding the 501 amino acid residues of the entire protein. cDNA 6.1 (189 bp) appears to encode the last 24 amino acids. Comparative amino acid sequence analysis indicated that P450 PCN1, PCN2, and HLp from rat and man, were 70, 67, and 73% homologous, respectively, to P450 3c. According to the cytochrome P450 nomenclature, the P450 3c gene is termed P450IIIA4.Comparison of the nucleotide sequences indicated that cDNA 6.1 was 100% homologous to cDNA 4.1. However, whereas a poly(A) tract started 23 nucleotides after the AATAAA consensus sequence in cDNA 6.1, cDNA 4.1 had a 3′ untranslated region extending 101 bp beyond the polyadenylation signal, which lacked poly(A). This observation is consistent with the previous finding that both cDNA 4.1 and 6.1 hybridized with two distinct species of poly(A)RNA (1700 and 1850 bases) from rabbit liver. The extreme 3′-end 79-bp fragment of cDNA 4.1 therefore was isolated by subcloning in pUC12 (clone p18-RsaI) and used to probe Northern blots of poly(A)RNA from control and rifampicin-treated rabbit liver. In contrast to cDNA 4.1 and 6.1, p18-RsaI cDNA hybridized only with the largest (1850 bases) mRNA species. We conclude that rabbit liver contains two P450 3c mRNA species differing in the length of their 3′ untranslated r

ISSN:1044-5498    

DOI:10.1089/dna.1988.7.39

年代:1988

数据来源: MAL

摘要:

ABSTRACTA HeLa cell nuclear transcription extract that is approximately 20 times more efficient than standard Hela cell transcription extracts was developed. Transcription of the strong adenovirus II major late promoter by this extract results in the synthesis of 1.5–4 molecules of product RNA per molecule of template, indicating that the extract is capable of multiple rounds of initiation. Standard HeLa cell nuclear extracts transcribe closed circular and linear adenovirus major late promoter templates with equal efficiency. In contrast, the new extract exhibits an increase of approximately twofold on transcription of a closed circular, as opposed to a linear, major late promoter templat

ISSN:1044-5498    

DOI:10.1089/dna.1988.7.47

年代:1988

数据来源: MAL

摘要:

ABSTRACTWe have found that 50 mML-histidine pH 7.6 as a buffer for gel electrophoresis greatly improves the resolution of oligonucleotides less than 70 residues long on denaturing polyacrylamide gels. The histidine buffer increases spacing between DNA bands on the gel about twofold in comparison with a standard buffer (89 mMTris-borate, 2 mMEDTA pH 8.3). In addition, low conductivity of the histidine buffer results in a threefold reduction of the electrophoresis time. Conditions for electrophoresis were optimized by varying both histidine and acrylamide concentrations. Other polycationic compounds, such as spermidine and ethylenediamine, were also tested for improved resolution of oligonucleotides. Several hypotheses as to the factors influencing the separation of DNA on gels are presented.

ISSN:1044-5498    

DOI:10.1089/dna.1988.7.57

年代:1988

数据来源: MAL

摘要:

ABSTRACTWe have evaluatedin vitroDNA amplification by polymerase chain reaction using either T4 DNA polymerase or Klenow fragment ofEscherichia coliDNA polymerase I. Both polymerases under optimal salt conditions permit efficient amplification of exon 3 of the hypoxanthine guanine phosphoribosyltransferase(HPRT)gene from human genomic DNA and from plasmid containing theHPRTcDNA. DNA sequences amplified from human genomic DNA, using two 20-nucleotide primers flanking the ends of the exon, showed a marked difference between the two polymerases. T4 DNA polymerase yielded only the expected amplified DNA fragment, whereas Klenow fragment produced many lower-molecular-weight bands in addition to the expected DNA fragment. On the basis of the reported fidelity ofin vitroDNA synthesis using Klenow fragment and T4 DNA polymerase, it is expected that the latter will create substantially fewer errors during the amplification process. For these reasons, T4 DNA polymerase should be particularly valuable for amplification of sequences present at a very low frequency requiring many cycles of amplification to be detected.

ISSN:1044-5498    

DOI:10.1089/dna.1988.7.63

年代:1988

数据来源: MAL