Biomedical subjects
Publications and source records attributed to S L Hu.
Biotin has been converted to 2-(biotinylamido)ethanol and condensed to phosphorylated oligonucleotides in a solid phase synthesis. The 5'-biotinylated oligonucleotides were enzymatically coupled to other DNA fragments by T4 DNA ligase or T4 RNA ligase. The hybridization properties of such biotin-labeled oligonucleotide probes were studied.
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Several reagents that react with sulfhydryl and amino groups were applied to voltage-clamped single nerve fibres of the frog. The fibres were exposed to comparable amounts of the chemical reagents for relatively short times. 3-(p-Hydroxyphenyl)propionic acid N-hydroxysuccinimide ester (HPPS), a substance which preferentially modifies amino groups, irreversibly reduced the size of the sodium and potassium current. The effect of HPPS on the Na current could be removed only partially by hyperpolarizing prepulses. N-ethylmaleimide (NEM), a reagent that preferentially reacts with sulfhydryl groups produced a small decrease of the sodium current which was removed almost completely by hyperpolarizing prepulses. NEM and HPPS shifted the voltage dependence of sodium inactivation, h infinity (E), to more negative values of membrane potential, but had little effect on the time course of sodium activation and inactivation. Pretreatment of a fibre with NEM did not prevent the action of HPPS; however, pretreatment of a fibre with HPPS decreased considerably the shift of the h infinity (E) curve caused by NEM. Our results suggest that modification of membrane bound amino groups affects the size of the ionic currents and the inactivation process. Although reagents that react with sulfhydryl groups were found to affect channel function, no definite evidence for the presence or absence of a functionally important sulfhydryl group on sodium channels has been obtained.
Hemagglutination activity was demonstrated in a Hantaan virus antigen prepared from infected suckling mouse brain by sucrose-acetone extraction. Hemagglutination of goose erythrocytes was pH dependent and was optimal at pH 6.0-6.2. Immunofluorescent and hemagglutination-inhibiting antibodies were significantly correlated in 80 human and 13 animal sera. Hemagglutination-inhibiting antibody was present in some patients 20 years after the onset of hemorrhagic fever with renal syndrome. The hemagglutination inhibition test was useful for confirming that an insectivore, Suncus murinus, may be an animal reservoir host of a Hantaan-like virus.
Cross-hybridization has been detected between corresponding regions of the genomes of bovine adenovirus type 3 and human adenovirus type 2. The most conserved region on the viral genomes encodes the hexon polypeptide. The nucleotide sequence of this region in bovine adenovirus type 3 has been determined. Comparison of the predicted amino acid sequences of the bovine adenovirus type 3 and human adenovirus type 2 hexon polypeptides reveals three regions of nonhomology.
We located the cleavage sites for restriction endonucleases EcoRI, HindIII, and BamHI on the genome of bovine adenovirus 7. Cross-hybridization at reduced stringency revealed two regions of homology shared by the DNA of human adenovirus 2 and bovine adenoviruses 7 and 3. These regions correspond to the hexon and the IVa2 protein genes of the human adenovirus. Another region of homology shared only by the human adenovirus and bovine adenovirus 7 corresponded to the penton or the polypeptide IIIa genes. These results allowed us to align the restriction map of bovine adenovirus 7 with respect to the other adenoviruses.
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We have constructed four novel adenovirus-SV40 hybrid viruses that contain the SV40 A gene at different positions downstream from the adenoviral major late promoter, within the region that encodes the second and third segments of the late tripartite RNA leader. The SV40 insert was precisely positioned in preselected regions of the adenoviral genome by using a combination of in vitro and in vivo recombination. As expected, all four recombinants produce equally high levels of SV40-encoded RNA that initiates at the adenovirus late promoter and contains two or three leader segments at the 5' end. Yet, in spite of this efficient transcription, only one virus, Ad-SVR284, directs the synthesis of high levels of SV40 large T antigen in infected cells; the other recombinants all produce approximately 20-fold less T antigen. This differential expression is, however, not seen in vitro, where equal amounts of hybrid T mRNA direct the synthesis of equal amounts of SV40 T antigen. Thus, some form of translational regulation is present in adenovirus-infected cells that is missing from the in vitro translation reaction.
We have identified a region of the viral genome required for the initiation of transcription in vitro from the major late promoter of adenovirus 2. A fragment of the adenovirus genome containing the cap site of the major late transcripts was inserted into plasmid pBR322 and cloned. Deletions were then generated in vitro in and around the T-A-T-A-A-A-A sequence located 25-31 nucleotides (positions -25 to -31) upstream from the cap site. DNAs with these deletions were tested for their ability to initiate transcription in vitro by the method of Manley et al. [Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3855-3859]. Whereas removal of sequences upstream from position -47 or downstream from position -12 did not abolish transcription, deletions extending into, or beyond, the T-A-T-A-A-A-A sequence reduced transcription to less than 1/10th. Removal of the normal cap site slightly reduced, but did not abolish, transcription. These results indicate that the region of the genome upstream of the cap site, with boundaries within 15-17 nucleotides to either side of the T-A-T-A-A-A-A sequence, is required for the initiation of transcription in vitro from the major late promoter of adenovirus 2.
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We have cloned and propagated in prokaryotic vectors the viral DNA sequences that are integrated in a variety of cells transformed by adenovirus 2 or SV40. Analysis of the clones reveals that the viral DNA sequences sometimes are arranged in a simple fashion, collinear with the viral genome; in other cell lines there are complex arrangements of viral sequences in which tracts of the viral genome are inverted with respect to each other. In several cases the nucleotide sequences at the joints between cell and viral sequences have been determined: usually there is a sharp transition between cellular and viral DNAs. The viral sequences are integrated at different locations within the genomes of different cell lines; likewise there is no specific site on the viral genomes at which integration occurs. Sometimes the viral sequences are integrated within repetitive cellular DNA, and sometimes within unique sequences. In some cases there is evidence that the viral sequences along with the flanking cell DNA have been amplified after integration. The sequences that flank the viral insertion in the line of SV40-transformed rat cells known as 14B have been used as probes to isolate, from untransformed rat cells, clones that carry the region of the chromosome in which integration occurred. Analysis of the structure of these clones by restriction endonculease digestion and heteroduplex formation shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration.
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