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Biomedical subjects

B H Howard

Publications and source records attributed to B H Howard.

At least 55 records · Page 3Linked to original sources
PubMed1990

DNA mutagenesis and recombination.

The polymerase chain reaction is used for site-specific mutagenesis and for DNA recombination without any enzymatic reaction in vitro, apart from DNA amplification.

DNA, Circular↗
PubMed1990

Transient analysis for antiproliferative gene activity.

A subset of tumor suppressor genes presumably functions by the inhibition of cellular proliferation; however, antiproliferative activity after transfection with putative suppressor genes has been difficult to demonstrate and often requires lengthy selection either in nude mice or in vitro. A rapid alternative is presented here that utilizes a gene encoding a surface marker protein to identify transfectants in a transient expression assay. In this assay the labeling index, rate of DNA synthesis, cell-cycle distribution, and surface receptor display are measured by flow cytometry. Human beta-interferon, a gene with proven antiproliferative activity, was studied using the transient analysis system. The beta-interferon gene was introduced into human tumor cells along with the marker gene encoding the 55-kDa subunit of the human interleukin 2 receptor. Within a few days of transfection, analysis of transfectants by flow cytometry revealed a decrease in the fraction of cells in G2/M and an increase in the fraction of cells in G1/G0 and S phases. The distortion of the cell cycle was accompanied by as much as a 69% reduction in the rate of DNA synthesis and, in some experiments, an unanticipated increase in the labeling index. Therefore, cells accumulating in S phase were not blocked but continued to synthesize DNA although at a reduced rate. These studies on DNA synthetic rates revealed the caveat that screening for antiproliferative candidate genes with a labeling index alone could, in certain circumstances, exclude potentially interesting sequences from further consideration. Although this transient analysis system was developed for studies on cellular proliferation, it may prove suitable for phenotypic assays on other genes as well.

Bromodeoxyuridine↗
PubMed1990

Alu interspersed repeats: selfish DNA or a functional gene family?

Within the genomes of higher eukaryotic cells, short interspersed repetitive sequences appear to be ubiquitous, but also remarkably varied with respect to copy number and position. Many of these repeat families, including the human Alu family, can be transcribed by RNA polymerase III, and evidence has accumulated from a variety of sources that levels of repeat transcripts whose transcription is dependent on RNA polymerase III are sensitive to cellular transformation as well as changes in differentiation state. Although interspersed repetitive sequences have in the past been dismissed as nonfunctional, the discovery of the linkage to differentiation state, as well as other recent developments, suggest that the question of repeat sequence functionality should be reexamined.

Animals↗
PubMed1990

A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles.

Site-specific mutagenesis and directional subcloning were accomplished by using the polymerase chain reaction to generate products that can recombine to form circular DNA. This DNA was transfected into E. coli without phosphorylation of primers, restriction enzyme digestion or ligation. Specifically, the polymerase chain reaction was used to generate products that when combined, denatured and reannealed, form double-stranded DNA with discrete, cohesive single-stranded ends. The generation of these cohesive ends of DNA permits the formation of precise, directional DNA joints without dependence on enzyme restriction sites. The primers were designed such that these cohesive single-stranded ends annealed to form circular DNA. The recombinant of interest was generated following only 14 amplification cycles. These recombinant circles of DNA were directly transfected into E. coli. In the mutagenesis protocol, the desired mutant was obtained at 83%-100% efficiency. Unwanted mutations were not detected, indicating a less than 0.025% nucleotide misincorporation frequency. In the directional subcloning protocol, inserts were positioned precisely in the recipient plasmid and were in the correct orientation. One unwanted mutation was detected after sequencing 900 bases, indicating a 0.11% nucleotide misincorporation frequency. Each manipulation, from setting up for the DNA amplification to transfection into E. coli. can easily be accomplished in one day.

Base Sequence↗
PubMed1989

Enhanced transfection efficiency and improved cell survival after electroporation of G2/M-synchronized cells and treatment with sodium butyrate.

To achieve high transfection efficiency in human fibroblasts with good preservation of proliferative capacity we developed an electroporation procedure that combines two distinct modalities: use of recipient cells synchronized in the late G2/mitotic phase of the cell cycle and treatment of cells post-electroporation with 5 mM butyrate. This combination enabled reduction of plasmid DNA concentration and electroporation voltage, both associated with cytotoxicity, while greatly enhancing transfection efficiencies. Although the method was primarily developed for transient expression it was also found to improve stable expression. This procedure should have wide applicability, particularly in studies seeking to identify DNA sequences that lead to inhibition of DNA synthesis and proliferation in human fibroblasts and other cells refractory to transfection.

Butyrates↗
PubMed1989

Efficient gene transfer by sequential treatment of mammalian cells with DEAE-dextran and deoxyribonucleic acid.

A variation of the classical DEAE-dextran method of gene transfer was developed for efficient transfection of HeLa cells with plasmid DNA. A brief exposure of the cells to medium containing DEAE-dextran was found to be sufficient for subsequent uptake of pRSVcat and to be superior to cocultivation of the cells with DEAE-dextran plus DNA. This sequential method of gene transfer is nontoxic and yielded up to 60% of HeLa cells positive for a surface protein encoded by the transfected sequence. The implications of this sequential transfection technique regarding the mechanisms of gene transfer are discussed.

Animals↗
PubMed1989

Studies on the molecular-genetic basis of replicative senescence in Werner syndrome and normal fibroblasts.

Based on evidence that human diploid fibroblasts (HDF) from the Werner syndrome (WS) of premature aging might overexpress an inhibitor of DNA synthesis (IDS), we prepared a eukaryotic cDNA expression library from WS mRNA and tested it for IDS activity in a transient assay. Two of six WS cDNA pools tested gave IDS activity, then on plus/minus screening revealed several differentially expressed cDNA clones. By slot blot and Northern analysis, one cDNA clone was found to be overexpressed in WS and normal senescent HDF, but not in quiescent normal HDF, indicating that it is senescence-specific. Further studies are needed to clarify: a) whether this cDNA truly acts as an IDS; b) if so, whether it acts alone or in concert with other cDNAs; and c) whether it is involved in the degenerative and malignant sequelae of WS and normal aging.

Aging↗
PubMed1988

Purification of transiently transfected cells by magnetic affinity cell sorting.

A method was developed to purify transiently transfected HeLa cells or African green monkey kidney CV-1 cells by magnetic affinity cell sorting. Monolayer cultures were transfected with mammalian expression vectors coding for either of two novel cell surface antigens, the Tac subunit of the human IL-2 receptor or vesicular stomatitis virus G protein. During the transient expression phase, cell populations were placed in suspension and mixed with monoclonal-antibody-coated magnetic particles in the presence of a sorting solution designed to minimize nonspecific cell/cell and cell/particle interactions. Transfected cells expressing the vector-encoded cell surface antigen were then isolated by application of a magnetic field. Reconstruction experiments indicated that IL-2 receptor-positive cells were bound about 100-fold more efficiently than receptor-negative cells. In transient transfection experiments, populations of greater than 90% antigen-positive cells were reproducibly obtained.

Animals↗
PubMed1988

Regulation of the CD2 alternate pathway of T cell activation by CD3. Evidence for heterologous desensitization.

Normal resting T cells were stimulated through the alternate CD2 pathway. A CD3 mAb VIT3 completely blocked their proliferative response. The time interval for 50% inhibition lasted for 24 h after the onset of CD2 stimulation. Mitogen-activated cloned long term cultured T cells could also be stimulated via CD2. This proliferative response was again inhibitable by VIT3, indicating that CD3 regulates the CD2 pathway not only in resting cells, but also in lymphocytes actively involved in an Ir. T cells were further loaded with Quin2 and their free cytoplasmic Ca2+ levels were monitored in response to CD3 and CD2 stimulation. Antibodies directed against both surface R triggered a rapid elevation of Ca2+ levels. Both responses were abrogated when the cells had been treated overnight with VIT3. The free cytoplasmic Ca2+ levels of VIT3-pretreated cells, however, were not higher than those of control cells. These results point to a functional interaction between CD3 and CD2 possibly at the level of signal transducing proteins. Finally, cholera toxin was found to inhibit the Ca2+ response in Jurkat T cells. Both the CD3 and CD2 stimulation were sensitive to cholera toxin, indicating that a GTP-binding protein may be involved in signal transduction for both surface structures.

Antibodies, Monoclonal↗
PubMed1987

Specific growth inhibitory sequences in genomic DNA from quiescent human embryo fibroblasts.

We used HeLa cells as recipients in a gene transfer assay to characterize DNA sequences that negatively regulate mammalian cell growth. In this assay, genomic DNA from quiescent human embryo fibroblasts was more inhibitory for HeLa replication than was DNA from either Escherichia coli or HeLa cells. Surprisingly, growth inhibitory activity depended on the growth state of the cells from which genomic DNA was prepared; it was strongest in DNA prepared from serum-deprived, quiescent embryo fibroblasts. This latter observation implies a role for DNA modification(s) in regulating the activity of the inhibitory sequences detected in our assay. The level of the observed growth inhibitory activity was sometimes high, suggesting that the relevant sequences may be abundantly represented in the mammalian genome. We speculate that these findings may provide new insights into the molecular mechanisms involved in cellular quiescence and in vitro senescence.

Cell Differentiation↗
PubMed1986

Detection of transcription and translation in situ with biotinylated molecular probes in cells transfected with recombinant DNA plasmids.

The efficiency of transfection and subsequent expression of recombinant DNA plasmids in monolayers of CV-1 monkey kidney cells was analyzed by immunoperoxidase and in situ hybridization with biotin-nucleotide-labeled DNA molecular probes. Two recombinant plasmids were used for transfection. Both contained the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV) as the transcriptional promoter, but two different coding sequences were employed [bacterial chloramphenicol acetyltransferase (pRSVcat) and mouse casein alpha (pRSVcsn alpha)]. In our experiments up to 25% of the transfected cells were positive for pRSVcat expression by indirect immunoperoxidase assay with affinity-purified, biotinylated anti-goat gamma-globulin after exposure to goat anti-chloramphenicol acetyltransferase antibody. In duplicate cultures, where pRSVcat expression was monitored by in situ hybridization signal that was restricted to the cytoplasm in positive cells was identified as pRSVcat RNA by its sensitivity to alkali. Although transfection of CV-1 cells with pRSVcsn-alpha did not result in immunologically detectable alpha casein, greater than 14% of the cells possessed cytoplasmic RNA concentrations detectable by in situ hybridization. These observations provide comparative information on in situ hybridization and immunoperoxidase techniques. They further indicate that in situ hybridization can be used to evaluate the effectiveness of transfection with recombinant expression vectors.

Acetyltransferases↗
PubMed1986

Reconstitution of an operon from overlapping fragments: use of the lambda SV2 integrative cloning system.

We have used the lambda SV2 system [Howard and Gottesman. In Gluzman (Ed.), Eukaryotic Viral Vectors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 211-216; in Inouye, M. (Ed.) Experimental Manipulations of Gene Expression. Academic Press, New York, 1983, pp. 137-153] to reconstitute the Salmonella typhimurium his operon from overlapping fragments. lambda SV2 can be propagated as an autonomously replicating plasmid or as a prophage integrated in the Escherichia coli chromosome at the lambda attachment site; our reconstitution was accomplished in the integrated state. We first inserted a portion of the his operon into lambda SV2 and integrated the resulting plasmid by site-specific recombination into the E. coli chromosome. This was achieved by brief induction of a resident prophage. The lysogen was then transformed with DNA from a lambda SV2 clone carrying the remainder of the his operon on an overlapping DNA fragment. The second plasmid was forced to integrate into the first by homologous recombination. When this recombination occurs at the his overlap, a lysogen carrying two lambda SV2 prophages is produced. One prophage carries the entire his operon and the other carries the his overlap region. The latter is removed by site-specific recombination, permitting further contiguous sequences to be sequentially added to the remaining prophage. This method should be applicable for the reconstitution and maintenance of large genes or gene clusters in the E. coli genome.

Alleles↗