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R Wu

Publications and source records attributed to R Wu.

At least 433 records · Page 24Linked to original sources
PubMed1982

Transcription terminates at lambda tR1 in three clusters.

Transcription of the rightward operon of bacteriophage lambda terminates in the presence of rho factor at a region known as tR1, located downstream of the structural gene for the lytic repressor cro. We demonstrate by nuclease Sl mapping that transcription termination at lambda tR1 occurs in vitro over a stretch of 60 nucleotides. End points of transcripts are clustered in three distinct regions, which we refer to as termination sites I, II, and III. Termination at site I is inhibited by L factor, whereas termination at sites II and III is not affected by L factor. The sensitivities of these sites to rho factor are in the order III greater than II greater than I. The cin-lcnc-l mutations abolish termination at site II but not at sites I and III; this result may explain the failure of these mutations to alleviate the phage requirement for N function for growth. Although possible stem-and-loop structures in the RNA transcript immediately upstream of each of the three termination sites can be found, no consistent correlation exists between the strengths of these stem-and-loop structures and the termination efficiencies of their respective sites.

Bacteriophage lambda↗
PubMed1982

Cytochrome c gene-related sequences in mammalian genomes.

We use a rat cytochrome c gene that we previously isolated and determined the sequence of to estimate the number of related sequences present in the rat genome. Approximately 25 different EcoRI restriction endonuclease fragments from total rat DNA hybridize to the gene of known structure. Four of these correspond to homologous sequences present in four different lambda Charon 4A-rat cytochrome c recombinants previously isolated. Intact or nearly intact genes appear to reside on almost all of the genomic fragments, because they hybridize strongly to gene subfragments representing both 5' and 3' portions of the coding sequence as well as to 3' noncoding DNA that is found specifically associated with the coding region. A subgroup of about six of the fragments also shares homology within the 73 nucleotides immediately preceding the AUG codon. An intron-specific probe reveals only the EcoRI fragment from which it was derived and one other genomic fragment. On the basis of the temperature of complete dissociation of the coding region probe in 0.75 M NaCl/0.075 M Na3 citrate/50% (vol/vol) formamide, the 25 fragments are separable into three stringency classes of 40-50 degrees C, 50-55 degrees C, and 55-60 degrees C. The latter, high-stringency group of about seven fragments includes those cloned in the recombinant phage isolates, whose regions homologous to cytochrome c are shown to differ from the purified gene of known sequence by an amount equivalent to about 2% mismatched bases. Families of cytochrome c gene-related sequences are also found in the genomes of several other mammals, including humans.

Animals↗
PubMed1982

Molecular cloning of the endogenous rat C-type helper virus DNA sequence: structural organization and functional analysis of some restricted DNA fragments.

Recently, we have identified and purified the integrated and proviral DNA sequences specific for two endogenous rat type C leukaemia helper viruses: WR-RaLV which originated from a fibrosarcoma induced in a feral rat and RHHV from the cell line HTC-H1 which originated from a Buffalo rat hepatoma. The rat leukaemia helper virus DNA sequences have previously been shown to be 8.4 to 8.8 kilobases (kb) in size. In this communication, we report the molecular cloning of the 8.8 kb DNA of RHHV by ligation at the BamHI site of the vector pBR322, cultured in an Escherichia coli RR1 host. After screening 5750 clones for ampicillin resistance and tetracycline sensitivity and testing by colony hybridization using 32P-labelled RHHV cDNA, four clones were isolated, two of which carried the total 8.8 kb DNA. A detailed restriction endonuclease map of the cloned RHHV DNA was deduced by sequential digestions of either 3'- or 5'-labelled DNA. Of the 14 restriction enzymes tested, EcoRI, BamHI, PstI, KpnI, TaqI, PvuII and SmaI gave informative cleavage patterns. At least two copies of long terminal repeated sequences (LTR) flanking the 3' and 5' termini of the proviral DNA were identified by TaqI and PstI cleavages. LTR in the rat endogenous leukaemia helper virus DNA measured 780 +/- 20 nucleotides in length. The genetic information encoded by the cloned DNA was also analysed by hybridization selection of RHHV mRNA, which was then used in cell-free protein synthesis in a rabbit reticulocyte lysate system. Essentially all major RaLV-specific proteins precipitable by anti-RaLV serum were synthesized in vitro, confirming that the RHHV genomic DNA was successfully cloned with little fidelity loss or scrambling of the genetic information.

Animals↗
PubMed1981

An improved procedure for utilizing terminal transferase to add homopolymers to the 3' termini of DNA.

Terminal deoxynucleotidyl transferase (E.C.2.7.7.3.1.) from calf thymus was used to add homopolymer tails to duplex DNA with 3' protruding, even, or 3' recessive ends. A gel electrophoresis method was employed to analyze the tail length and the percent of DNA with tails. In all the tailing reactions, dA, dT, and dC tails from CoCl2-containing buffer were longer than those from MnCl2 - or MgCl2 - containing buffers, whereas dG tails from MnCl2 -containing buffer were the longest. By varying the ratio of dNTP over DNA terminus and the concentration of terminal transferase, optimal conditions were found for adding dG or dC tails of 10-25 nucleotides in length and dA and dT tails of 20-40 nucleotides in length to duplex DNA with all types of 3' termini.

Animals↗
PubMed1981

Isolation and structure of a rat cytochrome c gene.

We screened a Charon 4A-rat genomic library using the cloned iso-1 cytochrome c gene from Saccharomyces cerevisiae as a specific hybridization probe. Eight different recombinant phages homologous to a coding region subfragment of the yeast gene were isolated. Nucleotide sequence analysis of a 0.96-kilobase portion of one of these established the existence of a gene coding for a cytochrome c identical in amino acid sequence with that of mouse. The rat polypeptide chain sequence had not previously been determined. In contrast to the yeast iso-1 and iso-2 cytochrome c genes, neither of which have introns, the rat gene contains a single 105-base pair intervening sequence interrupting glycine codon 56. The overall nucleotide sequence homology between cytochrome c genes of yeast and rat is about 62%, with areas of greater homology coinciding with four regions of functionally constrained amino acid sequences. Two of these regions displayed 85-90% DNA sequence homology, including the longest consecutive homologous stretch of 14 nucleotides, corresponding to amino acids 47-52 of the rat protein. Somewhat less homology was observed in the DNA-specifying amino acids 70-80, which are invariant residues in most known cytochrome c molecules. Thermal dissociation of the yeast probe from the homologous rat DNA was at about 58 degrees C in 0.39 M Na+. These results establish that cytochrome c genes may be isolated by interspecies hybridization between widely divergent organisms.

Amino Acid Sequence↗
PubMed1981

Distinctive effects of hydrocortisone on the modulation of EGF binding and cell growth in HeLa cells grown in defined medium.

Hydrocortisone modulates the binding capacity of HeLa cells for 125I-labeled epidermal growth factor (EGF). A twofold increase in 125I-labeled EGF binding is observed within 24 hours after the addition of pharmacological concentration of hydrocortisone (5 X 10(-8) - 1 X 10(-6) M). This enhancement of binding is reversible, and occurs when the cells are cultured in either serum-supplemented or completely defined, serum-free, hormone-supplemented medium. Scatchard analysis of the binding data indicates that the number of 125I-EGF binding sites is increased, and that no appreciable change in the affinity of the EGF receptor for labeled EGF occurs. In the serum-free condition hydrocortisone stimulates the growth of HeLa cells, but we have observed no connection between this growth stimulation and the enhancement of EGF binding. The growth response to hydrocortisone is independent of EGF, and the concentration dependency of the growth response to EGF is unaltered by the addition of hydrocortisone to the medium. Hydrocortisone elicits the growth response at a concentration as low as 5 X 10(-9) M, while a concentration higher than 5 X 10(-6) M is required to affect the binding capacity for 125-EGF. These effects are specific for glucocorticoid steroids. Similar concentrations of progesterone, testosterone, or estradiol produce no measurable response. Although the elevation of EGF receptor levels in the serum-supplemented medium is similar to that observed in the serum-free cultures, hydrocortisone is growth-inhibitory under these conditions. This growth inhibition occurs at pharmacological concentrations of hydrocortisone with a concentration dependency that is similar to that of the EGF receptor modulation.

Blood↗
PubMed1981

DNA sequence of a mutation in the leader region of the yeast iso-1-cytochrome c mRNA.

We have constructed a plasmid that selectively integrates adjacent to the CYC1 locus, which determines iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. Different CYC1 alleles can be conveniently recovered by digestion of total DNA from transformed strains with BgI II, a restriction endonuclease that does not cut the vector or the CYC1 gene, followed by transformation of Escherichia coli, selecting the ampicillin resistance gene carried on the original vector. This procedure was used to clone the cyc1-362 gene, which contains an alteration in front of the AUG initiation codon. The cyc1-362 mutational causes a deficiency of the iso-1-cytochrome c protein but still allows transcription of the iso-1-cytochrome c mRNA. DNA sequence analysis showed that the cyc1-362 mutation consisted of two single-base-pair substitutions, producing an A leads to G change 18 nucleotides and a G leads to A change 30 nucleotides in front of the AUG initiation codon in the mRNA. The A leads to G change at position -18 resulted in the creation of an AUG triplet, which is proximal to the normal initiation site and out of phase with the normal reading frame. The deficiency of iso-1-cytochrome c is most simply explained by assuming that translation initiates at the more proximal abnormal AUG site but not at the normal AUG site.

Base Sequence↗
PubMed1981

Modification of the bacteriophage vector M13mp2: introduction of new restriction sites for cloning.

The construction of two new derivatives of the bacteriophage cloning vector M13mp2 is described. One derivative, mWJ22, contains a new HindIII site while the other, mWJ43, contains a new BamHI site. These new sites were both introduced at the EcoRI site at amino acid five of the 145 amino acid-long fragment of Escherichia coli beta-galactosidase within the phage. The new restriction sites do not disrupt the blue color detection system of M13mp2; therefore insertion of cloned fragments results in colorless plaques on indicator plates for the new derivatives.

Base Sequence↗
PubMed1981

Abnormal hypothalamic-pituitary function in polyostotic fibrous dysplasia.

Hypothalamic-pituitary dysfunction was found in two patients with polyostotic fibrous dysplasia and leontiasis ossea. Both patients probably had McCune--Albright syndrome with early sexual development, disfiguring craniofacial bone lesions, cutaneous hyperpigmentation and gigantism in childhood. Endocrinological studies revealed measurable plasma growth hormone throughout a 24-h sampling period with preservation of sleep augmented rises. The mean 24-h values were 329 and 7 ng/ml, respectively. Both their mean plasma prolactin concentrations, 385 and 45 ng/ml, and the 24-h secretory patterns of prolactin were abnormal. One patient had an elevated mean plasma cortisol concentration of 8.5 micrograms/dl but the 24-h pattern of cortisol secretion was normal. Basal plasma LH, FSH, TSH, T4 and T3 concentrations were normal but neither patient had a TSH response to TRH. Prolactin and growth hormone secretions were, however, increased after TRH. Oral glucose resulted in partial suppression of GH but an exaggerated insulin response in both patients. Post-mortem in one patient revealed a thick calvarium with bony encasement of the pituitary gland. The pituitary and hypothalamus appeared normal on gross, light microscopic and electronmicroscopic examination. These data strongly suggest the presence of hypothalamic-pituitary dysfunction in these two patients.

Adult↗
PubMed1980

Synthesis of the human insulin gene. Part III. Chemical synthesis of 5'-phosphomonoester group containing deoxyribooligonucleotides by the modified phosphotriester method. Its application in the synthesis of seventeen fragments constituting human insulin C-chain DNA.

A method for phosphorylating a protected deoxyribooligonucleotide containing phosphotriester linkages is described. The modified phosphotriester method of chemical synthesis is further refined in terms of (i) better final deblocking conditions and (ii) new chromatography solvent systems containing acetone-water-ethyl acetate to yield pure oligomers. The effectiveness of these improvements has been demonstrated in the rapid and efficient synthesis of seventeen fragments constituting the sequence of human insulin C-chain DNA.

Base Sequence↗
PubMed1980

Unequal crossing over in the ribosomal DNA of Saccharomyces cerevisiae.

Unequal sister chromatid exchanges occur at the ribosomal DNA locus of yeast during mitotic growth. The frequency of unequal crossing over, as measured by the deletion or duplication of an inserted genetic marker (LEU2), is sufficient to maintain the sequence homogeneity of the rDNA repeat units.

Chromosome Deletion↗
PubMed1980

Epidermal growth factor-induced down-regulation of receptor does not occur in HeLa cells grown in defined medium.

125I-Labeled epidermal growth factor (EGF) binds to HeLa cells in culture under completely defined conditions in a specific and saturable manner. When the cultures are maintained in serum, all the reported phenomena of down-regulation occur. In contrast, in cultures maintained in serum-free hormone-supplemented medium, binding is maximal after 50 min and remains constant for at least 18 hr. EGF retains its mitogenic activity in this defined system; as little as 4 pM EGF elicits a 2-fold increase in cell number in 5 days. EGF appears to be in reversible equilibrium with a constant number of surface receptors, and gel filtration analysis indicates a decrease in the rate of degradation and release of bound 125I-labeled EGF. These results indicate that EGF can retain its mitogenic activity in the absence of concomitant down-regulation.

Cell Division↗
PubMed1980

BK virus DNA sequence coding for the t and T antigens and evaluation of methods for determining sequence homology.

The DNA sequence of the early region of the human papovavirus BK (MM strain) was determined. A potential initiation signal for translation is located at nucleotides 3,047 to 3,045 or map position 0.614. Extending counterclockwise from this AUG signal there is only one open reading frame, which can code for a putative t antigen of 100 amino acids in length. If the early mRNA of BKV is spliced, then the regions between nucleotides 3,047 to 2,808 and 2,725 to 884 can code for a T antigen 694 amino acids in length. The sequences of the deduced T antigens in BK virus share 71% amino acid homology with those in simian virus 40, whereas the coding sequences of the two viruses share 70% DNA homology. Comparison of DNA sequences and evaluation of homology measurements between these two viruses are discussed.

Antigens, Neoplasm↗
PubMed1980

Synthesis of the human insulin gene. Part IV. New synthetic deoxyribooligonucleotide adaptors and primer for DNA cloning and sequence analysis.

The chemical synthesis of four new deoxyribooligonucleotides to be used as adaptors in molecular cloning of DNA for expression studies is described. These are (i) start-adaptor, (ii) stop-adaptor, (iii) conversion adaptor to insert ribosomal binding site, and (iv) retrieving adaptor. We have also synthesized a 19-bases-long primer, 5'-TTGTAAAACGACGGCCAGT-3' to increase the speed of DNA sequence analysis and also demonstrated its application using single-stranded M13 DNa vector.

Base Composition↗
PubMed1979

Synthesis of the human insulin gene. Part II. Further improvements in the modified phosphotriester method and the synthesis of seventeen deoxyribooligonucleotide fragments constituting human insulin chains B and mini-CDNA.

The purification of protected deoxyribooligonucleotides containing phosphotriester internucleotidic linkages has been improved by developing a deactivated silica gel chromatographic technique. The efficiency of this technique as applied in the modified phosphotriester approach has been demonstrated in the rapid synthesis of seventeen pure fragments constituting the sequence of human insulin B and mini-C DNA. The sequence of each oligomer was confirmed by the two-dimensional mobility shift method of fingerprinting.

Base Sequence↗