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Biomedical subjects

H V Huang

Publications and source records attributed to H V Huang.

32 records · Page 2Linked to original sources
PubMed1988

Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro.

Genetic loci affecting Sindbis virus pathogenesis in neonatal mice have been examined by using a full-length cDNA clone of the virus (Toto1101). The full-length cDNA is linked to a bacteriophage SP6 promoter to facilitate the synthesis of infectious RNA transcripts in vitro. Virus derived from Toto1101 showed reduced virulence (attenuation) in neonatal mice. Replacement of the E1 glycoprotein and 6K genes of Toto1101 with cloned E1 and 6K genes derived from a virulent Sindbis virus strain, AR339 (SB), resulted in a new construct, TR2000, that gave rise to virulent virus. Sequence determinations for the entire substituted regions of TR2000, Toto1101, and related virulent and attenuated strains identified three coding differences in E1 between Toto1101 and TR2000. These differences, individually or in combination, may be responsible for the attenuated phenotype. Previous studies in this laboratory identified another attenuating mutation at amino acid position 114 of the E2 glycoprotein (N.L. Davis, F.J. Fuller, W.G. Dougherty, R.A. Olmsted, and R.E. Johnston, Proc. Natl. Acad. Sci. USA 83:6771-6775, 1986). Substitution of Arg-114 in the mutant SB-RL for Ser-114 of SB appears to confer three distinguishing phenotypes: attenuation in neonatal mice, increased sensitivity to specific E2 monoclonal antibodies, and accelerated penetration of BHK cells. Replacement of TR2000 sequences containing the codon for amino acid 114 of E2 with corresponding fragments from cDNA clones of SB or SB-RL produced two strains of Sindbis virus (TR2100 and TR2200) which were isogenic except for the E2 114 codon (Ser and Arg, respectively). The three diagnostic phenotypes cosegregated according to the origin of the codon for amino acid 114 of E2, confirming the dramatic effect of this single amino acid substitution on these three phenotypes.

Amino Acid Sequence↗
PubMed1987

Structure of the T15 VH gene subfamily: identification of immunoglobulin gene promotor homologies.

We have sequenced and analyzed the flanking and coding regions of four closely related mouse VH gene segments referred to as the T15 gene subfamily. We have found that although the sequence homologies between the four members of this family are greatest in the coding region, significant homologies extend into the leader sequences and the flanking regions as well. Sequence comparisons of the promoter regions of other VH gene segments indicate that the octamer promoter sequence is part of a larger conserved sequence that may play an important role in immunoglobulin gene transcription. Analysis of the lambda clones containing the members of this subfamily indicate that despite the close linkage of two of the members, the average number of kilobases of DNA between the germ-line gene segments of this family is likely to be very great.

Animals↗
PubMed1987

Molecular dissection of the murine antibody response to streptococcal group A carbohydrate.

Monoclonal antibodies (mAb) to streptococcal group A carbohydrate (GAC) are encoded by a minimum of two VH, four JH, four V kappa, three J kappa, one V lambda, and one J lambda gene segments. The IdX, IdI-1, and Id5 idiotypic determinants are expressed by anti-GAC mAb and are found on free kappa chains. Each pattern of these determinants is encoded by a distinct V kappa gene segment, apparently without the requirement for a particular J kappa, VH, or JH gene segment, or somatic mutation. In contrast, the binding site-associated idiotypic determinant IdI-3a does not correlate with any single V or J gene segment.

Animals↗
PubMed1987

Efficient RecABC-dependent, homologous recombination between coliphage lambda and plasmids requires a phage ninR region gene.

A phage lambda gene that gives a 100-fold increase in recombinant frequencies for RecABC pathway-mediated, phage-plasmid homologous recombination (Shen and Huang 1986) maps to ninG (orf 204) of lambda. We call this gene rap, for recombination adept with plasmid. A similar determinant exists in Charon 4A and maps in phi 80-derived sequences, between nin5 and the Rz homology with lambda. The absence of the Rap+ phenotype from certain lambda vectors explains the inefficiency of screening the resulting phage libraries using phage-plasmid homologous recombination. The mapping of rap permits the construction of lambda vectors more suitable for this screening technique.

Bacteriophage lambda↗
PubMed1987

Syrinx 2A: an improved lambda phage vector designed for screening DNA libraries by recombination in vivo.

The Syrinx 2A phage and pi AN13 plasmid were designed for screening of DNA libraries by homologous recombination in vivo. Syrinx 2A carries multiple cloning sites and a recently identified lambda gene, rap (recombination adept with plasmid), required for efficient phage-plasmid recombination. We describe a rapid, reliable, and technically easy method to screen Syrinx 2A libraries, expand the resulting phage-plasmid cointegrates, and subclone plasmid in as little as 2 days. Recombination screening allows one specific member of a closely related multigene family to be isolated selectively.

Bacteriophage lambda↗
PubMed1987

Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants.

We constructed full-length cDNA clones of Sindbis virus that can be transcribed in vitro by SP6 RNA polymerase to produce infectious genome-length transcripts. Viruses produced from in vitro transcripts are identical to Sindbis virus and show strain-specific phenotypes reflecting the source of RNA used for cDNA synthesis. The cDNA clones were used to confirm the mapping of the causal mutation of ts2 to the capsid protein. A general strategy for mapping Sindbis virus mutations is described and was used to identify two lethal mutations in an original full-length construct which did not produce infectious transcripts. An XbaI linker was inserted in the cDNA clone near the transcriptional start of the subgenomic mRNA; the resulting virus retains the XbaI recognition sequence, thus providing formal evidence that viruses are derived from in vitro transcripts of cDNA clones. The potential applications of the cDNA clones are discussed.

Animals↗
PubMed1986

Homologous recombination in Escherichia coli: dependence on substrate length and homology.

We studied the in vivo recombination between homologous DNA sequences cloned in phage lambda and a pBR322-derived plasmid by assaying for the formation of phage-plasmid cointegrates by a single (or an odd number of) reciprocal exchange. (1) Recombination proceeds by the RecBC pathway in wild-type cells and by low levels of a RecF-dependent pathway in recBC- cells. The RecE pathway appears not to generate phage-plasmid cointegrates. (2) Recombination is linearly dependent on the length of the homologous sequences. In both RecBC and RecF-dependent pathways there is a minimal length, called the minimal efficient processing segment (MEPS), below which recombination becomes inefficient. The length of MEPS is between 23-27 base pairs (bp) and between 44-90 bp for the RecBC- and RecF-dependent pathways, respectively. A model, based on overlapping MEPS, of the correlation of genetic length with physical length is presented. The bases for the different MEPS length of the two pathways are discussed in relationship to the enzymes specific to each pathway. (3) The RecBC and the RecF-dependent pathways are each very sensitive to substrate homology. In wild-type E. coli, reduction of homology from 100% to 90% decreases recombinant frequency over 40-fold. The homology dependence of the RecBC and RecF-dependent pathways are similar. This suggests that a component common to both, probably recA, is responsible for the recognition of homology.

Bacteriophage lambda↗
PubMed1980

A computer simulation of evolutionary forces controlling the size of a multigene family.

A Monte Carlo-type simulation of the evolution of a multigene family was performed. The model was designed to study the selective forces which may control the size of a multigene family. As expected, we find that direct selection on the size of the multigene family can control its size. More important, we find that selection acting upon the family as a single functional unit, in conjunction with homologous but unequal crossing over, can also control the size of a multigene family.

Animals↗
PubMed1978

Bursectomy in ovo blocks the generation of immunoglobulin diversity.

Immunoglobulins made by chickens bursectomized in ovo on day 11 of incubation were studied by two-dimensional gel electrophoresis. All such bursectomized chickens have limited diversity in their immunoglobulin molecules. A range of different degrees of diversity restriction was found in individual bursectomized chickens. The limited immunoglobulin diversity was stable for at least 20 weeks in that the same spots were found over this time span. Bursectomized chickens that responded to repeated antigen challenge had sheep erythrocyte- and dinitrophenol-specific antibodies of limited diversity. Different bursectomized responders made almost identical antigen-specific antibodies. The results were interpreted as indicating that individual bursectomized birds had been blocked at different points during the developmental generation of immunoglobulin diversity, and that the genetic mechanisms that underlie the developmental diversification of clones of B cells lead to a sequential generation of very similar antibody molecules in different embryos.

Agammaglobulinemia↗
PubMed1977

The area-code hypothesis: the immune system provides clues to understanding the genetic and molecular basis of cell recognition during development.

Numberous studies of embryogenesis have provided evidence for highly specific cell-surface recognition phenomena. These include both the interactions of neighboring cells and the specific cellular migrations which occur as the developmental program of the embryo progresses. The area-code hypothesis elaborate here is an attempt to provide a framework for understanding cell-recognition phenomena in development. This hypothesis is based on extensive genetic, molecular, and cellular studies of the immune system. These studies suggest that the following events occur during the differentiation of antibody-producing cells. 1) Somatic cell lines of antibody-producing cells undergo a modification of their DNA as they become committed to synthesize a particular type of antibody molecule. This chromosomal modification event is probably a DNA translocation which leads to a somatic rearrangement of certain antibody genes. 2) In each of the specific cell lineages the new arrangement of DNA is inherited by all subsequent generations of cells. 3) The developmental programs which control these genetic alterations may be employed in a programmed and reproducible fashion. This programming of antibody development is suggested because different embryos appear to become committed to the production of identical antibody molecules in the same developmental sequence. 4) Antibody molecules are initially displayed on the cell surface where they serve as highly specifici receptors to trigger the cell to proliferate and differentiate upon interacting with appropriate external molecular signals. 5) Antibody-producing cells display combinations of different molecules on their surfaces which cause each of a very large number of different cells to interact differently with their environment. 6) The genes which code for many of these cell-surface molecules are organized into multigene families. These observations as well as information from other developmental systems have led us to propose the area-code hypothesis. This hypothesis is concerned with the structure, function, and regulation of cell-surface molecules that mediate recognition phenomena during embryogenesis. Area-code molecules are cell-surface molecules which are involved in the specific recognition phenomena during growth and development. These molecules provide cells with distinct cell-surface addresses or phenotypes, and provide the basis for the specificity in cell-cell recognition during cell migrations and cell-cell interactions, as well as serving as receptors for diffusible differentiation signals. The area-code hypothesis has 3 main postulates. i) There is a progressive display of specific combinations of area-code molecules on the surfaces of cells during development. ii) The genetic programs which determine the specific expression of area-code molecules are in part controlled by DNA modifications. These chromosomal modifications are believed to channel cells into specific lineages uith progressively restricted developmental options...

Animals↗
PubMed1975

Application of latex microspheres in the isolation of plasma membranes. Affinity density perturbation of erythrocyte membranes.

Immunolatex spheres, originally developed as visual markers for scanning electron microscopy, were employed as membrane density perturbation reagents. Methacrylate spheres were bound to antibody molecules and used to label antigens on erythrocytes. Ghosts prepared from labeled cells were subjected to isopycnic centrifugation on continuous sucrose and dextran gradients. It was found that the labeled erythrocyte membranes had a substantially higher density than unlabeled membranes. The extent to which the membrane density was shifted on a given gradient depended on the number, size and density of the latex spheres and could be closely predicted by theory. These results suggest that the reagents and techniques described here have potential application for the isolation of plasma membranes from more complex cell types.

Animals↗