ISSN:1044-5498    

DOI:10.1089/dna.1.1982.1.99

年代:1982

数据来源: MAL

ISSN:1044-5498    

DOI:10.1089/dna.1.1982.1.101

年代:1982

数据来源: MAL

ISSN:1044-5498    

DOI:10.1089/dna.1.1982.1.103

年代:1982

数据来源: MAL

摘要:

Thirty-fourEcoRI* sites have been identified on the nucleotide sequence of CaMV, following cloning ofEcoRI* fragments in M13mp2. From this sequencing data, we have deduced thatEcoRI* recognizes sites that differ in a single position from the canonicalEcoRI sequence, GAATTC. Any substitution can occur at any one of the six positions in the recognition site, with the exception of A→T or T→A changes within the central tetramer. TheEcoRI* restriction patterns of øx174 and pBR322 are consistent with these recognition criteria. Similarly,BamHI* cleavage of øx174 and SV40 (Georgeet al., 1980) produces restriction patterns that are consistent with single-position degeneracy in the canonicalBamHI recognition site. Cohesive termini produced byEcoRI* cleavage were ligated into theEcoRI site of M13mp2, even when there was a base pair mismatch within the four nucleotide overlap. Mismatches were corrected asymmetrically during subsequent replication of M13 inE.

ISSN:1044-5498    

DOI:10.1089/dna.1.1982.1.109

年代:1982

数据来源: MAL

摘要:

Under standard conditions,EcoRI endonuclease uniquely recognizes the inverted repeat GAATTC. However, this specificity breaks down under non-standard conditions into what has been termedEcoRI* specificity, wherein many other sequences are recognized. We show here that the hydrolysis rates at all knownEcoRI* sites can be summarized by the hierarchies: G>>A>T>>C at the first position, A>>[G, C]>>T at the second and third position, and the corresponding complements at the last three positions. This is consistent with a recognition model which assumes that there are two specific hydrogen bonds per base pair under standard conditions. One or more of these are randomly replaced by water underEcoRI* conditions and the position of a sequence within the appropriate hierarchy is primarily determined by the number of retained hydrogen bonds. These retained hydrogen bonds are common recognition features that can be identified by examining the DNA. The recognition points thereby identified forEcoRI all fall within the major groove of the DNA.

ISSN:1044-5498    

DOI:10.1089/dna.1.1982.1.117

年代:1982

数据来源: MAL

摘要:

Eleven chimeric plasmids have been constructed which direct the synthesis of mature human fibroblast (IFN-β1) or leukocyte interferon (IFN-αA) proteins under the control of theE. coli trppromoter. The plasmids differ with respect to the nucleotide spacing between the Shine-Dalgarno sequence of thetrpleader and theATGtranslation start signal of the interferon genes. By utilizing a uniqueXbaI endonuclease site located within the spacer region of the expression plasmids, the spacings were altered from 2-10 nucleotides or 7-15 nucleotides for the fibroblast and leukocyte interferon expression plasmids, respectively. The optimal spacing for expression, as determined by interferon assay, is 9 nucleotides for both types of transcripts, despite differences in nucleotide sequence within the spacer region and downstream from theAUGinitiator. Yields of IFN-αA varied about six-fold, while among the different IFN-β1expression plasmids a range of more than 100-fold in interferon production was observed. The difference in the range of variation between the IFN-αA and IFN-β1plasmids is attributed partly to changes in messenger RNA secondary structure within the ribosome binding sites which affect message half

ISSN:1044-5498    

DOI:10.1089/dna.1.1982.1.125

年代:1982

数据来源: MAL

摘要:

The human pro-opiomelanocortin (POMC) gene has been characterized by molecular cloning and DNA sequence analysis. Although this gene codes for several different polypeptide hormones, only a single intron interrupts the protein coding region. The DNA in this intron, and in a second intron found in the region of the gene homologous to the mRNA 5′-untranslated sequence, contains repetitive DNA sequences. At least some of these sequences belong to theAlufamily of transcribed middle repetitive DNA. The determination of the complete nucleotide sequence of the coding regions of the gene demonstrates that the pattern of homologous and variable regions seen in the POMC protein between different species is reflected at the DNA level. DNA sequences encoding the highly conserved regions of POMC are 90-95% homologous between species while the coding sequences for the variable regions of the protein are approximately 70% homologous. The very high degree of homology in the amino terminal portion of POMC is consistent with an important physiological role for this peptid

ISSN:1044-5498    

DOI:10.1089/dna.1.1982.1.133

年代:1982

数据来源: MAL

摘要:

Production of growth hormone by cultured rat pituitary tumor cells (GH3subline) is regulated by hormones. In previous studies, it was shown that thyroid and glucocorticoid hormones stimulate growth hormone synthesis. In the current studies, a cloned cDNA probe complementary to growth hormone mRNA was used to determine, by solution hybridization, the number of growth hormone mRNA molecules per cell under various hormonal conditions. The experiments were performed with cells grown in three different medium conditions to distinguish the effect of the added hormones from the effect of serum factors. The results show that, within 72 h, both thyroid and glucocorticoid hormones, when added alone, are able to induce a significant accumulation of cytoplasmic growth hormone mRNA. However, to obtain their maximal efficiencies, the two types of hormones have different requirements: glucocorticoids appear to need only the presence of thyroid hormones, whereas thyroid hormones appear to require some as yet unidentified serum factors. When added together, the two classes of hormones act synergistically. The maximal range of growth hormone mRNA in these cells varies by over 500-fold—from about 1000 molecules per cell (10% calf serum plus thyroid and glucocorticoid hormones) to less than two molecules per cell (in a defined serum-substitute-containing medium with no hormone added). The data further suggest that, in these GH3cells, the efficiency of translation of growth hormone mRNA can also vary (by a factor of 4), depending on the hormonal conditions used. However, these latter influences are not specific for growth hormone mRNA but rather appear to be a general translational effect on all the mRNA population. These results demonstrate that the major effect of thyroid and glucocorticoid hormones on growth hormone production is due to effects on the number of growth hormone mRNA molecule

ISSN:1044-5498    

DOI:10.1089/dna.1.1982.1.145

年代:1982

数据来源: MAL

摘要:

Relaxin is a peptide hormone produced by the corpora lutea of ovaries during pregnancy, softening and lengthening the ligaments of the pelvis and softening the cervix in order to make childbirth easier. In attempts to determine the nucleotide sequence coding for relaxin, recombinant DNA techniques were used to obtain a cDNA clone bank from total mRNA isolated from the ovaries of pigs in late pregnancy. Clones were screened using cDNA initiated by synthetic oligonucleotide primers coding for the Trp Val Glu Ile sequence of the porcine relaxin B chain. The synthetic undecamer [5′-ATCTCCACCCA-3′] was found to prime a specific32P-labeled cDNA of approximately 300 nucleotides containing B chain and signal peptide coding sequences, as verified by nucleic acid sequence analysis. This cDNA was used to probe the ovarian clone bank. Several clones containing large inserts which hybridized to this probe were subjected to sequence analysis and some of these were found to contain the preprorelaxin coding region, comprising a signal peptide of 24 amino acids, a B chain of 32 amino acids, a large C peptide of 104 amino acids, and an A chain of 22 amino acids. From the amino acid sequence of prorelaxin derived in this way, it appears that the processing of prorelaxin involves two enzymes with chymotrypsin-like and trypsin-like specificity, respectively. In comparisons of porcine and rat preprorelaxins, the C region had as much amino acid sequence homology as the B and A chains. The C region is also rich in charged amino acids, suggesting a role for it beyond simply ensuring proper disulfide bond format

ISSN:1044-5498    

DOI:10.1089/dna.1.1982.1.155

年代:1982

数据来源: MAL

ISSN:1044-5498    

DOI:10.1089/dna.1.1982.1.163

年代:1982

数据来源: MAL