Search PubMed⌕ Search

Biomedical subjects

L Hood

Publications and source records attributed to L Hood.

At least 109 records · Page 6Linked to original sources
PubMed1997

Inexpensive handheld device for the construction of high-density nucleic acid arrays.

We report an easy-to-use, 384-pin handheld arraying and replicating device (ARD) for constructing high-density replicas of nucleic acids and E. coli transformants. We have modified an existing 384-pin tool to include a novel guide system to ensure vertical pin movement and enhance reproducibility. An asymmetric rectangular multiplexing frame is designed to increase the array density to 1536 dots on a standard microplate-size nylon membrane and to reduce the time and effort involved in producing array replicas. Our initial studies used the ARD to construct 1536-dot arrays of ovarian cDNA clones. We have hybridized these arrays with 32P-labeled probes, which resulted in distinctive signals for either visual interpretation or semiautomatic spot detection and signal integration.

Autoradiography↗
PubMed1996

High-throughput plasmid mini preparations facilitated by micro-mixing.

We have developed a reliable high-throughput plasmid isolation system using a 96-well plate format. This system combines a novel glass bead micro-mixing method with modified alkaline lysis and Sephacryl S-500 DNA purification procedures. Mechanical forces generated by vortexing glass beads inside each well of the 96-well plates ensure that the bacterial pellets are homogeneously resuspended, the cells are completely lyzed, and the resulting bacterial lysates are thoroughly mixed with the potassium acetate solution. The vortexing speed and duration for glass bead mixing have been standardized to facilitate plasmid DNA yields without significant adjustments.

Cells, Cultured↗
PubMed1996

Random mutagenesis of Thermus aquaticus DNA polymerase I: concordance of immutable sites in vivo with the crystal structure.

Expression of Thermus aquaticus (Taq) DNA polymerase I (pol I) in Escherichia, coli complements the growth defect caused by a temperature-sensitive mutation in the host pol I. We replaced the nucleotide sequence encoding amino acids 659-671 of the O-helix of Taq DNA pol I, corresponding to the substrate binding site, with an oligonucleotide containing random nucleotides. Functional Taq pol I mutants were selected based on colony formation at the nonpermissive temperature. By using a library with 9% random substitutions at each of 39 positions, we identified 61 active Taq pol I mutants, each of which contained from one to four amino acid substitutions. Some amino acids, such as alanine-661 and threonine-664, were tolerant of several or even many diverse replacements. In contrast, no replacements or only conservative replacements were identified at arginine-659, lysine-663, and tyrosine-671. By using a library with totally random nucleotides at five different codons (arginine-659, arginine-660, lysine-663, phenylalanine-667, and glycine-668), we confirmed that arginine-659 and lysine-663 were immutable, and observed that only tyrosine substituted for phenylalanine-667. The two immutable residues and the two residues that tolerate only highly conservative replacements lie on the side of O-helix facing the incoming deoxynucleoside triphosphate, as determined by x-ray analysis. Thus, we offer a new approach to assess concordance of the active conformation of an enzyme, as interpreted from the crystal structure, with the active conformation inferred from in vivo function.

Amino Acid Sequence↗
PubMed1996

The complete 685-kilobase DNA sequence of the human beta T cell receptor locus.

The human beta T cell receptor (TCR) locus, comprising a complex family of genes, has been sequenced. The locus contains two types of coding elements--TCR elements (65 variable gene segments and two clusters of diversity, joining, and constant segments) and eight trypsinogen genes --that constitute 4.6 percent of the DNA. Genome-wide interspersed repeats and locus-specific repeats span 30 and 47 percent, respectively, of the 685-kilobase sequence. A comparison of the germline variable elements with their approximately 300 complementary DNA counterparts reveals marked differential patterns of variable gene expression, the importance of exonuclease activity in generating TCR diversity, and the predominant tendency for only functional variable elements to be present in complementary DNA libraries.

Amino Acid Sequence↗
PubMed1996

A new strategy for genome sequencing.

Existing approaches to sequencing the human genome are based on the assumption that each region to be sequenced must first be mapped. But there is a simpler strategy in which any number of laboratories can cooperate.

Animals↗
PubMed1996

Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen.

With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.

AIDS Vaccines↗
PubMed1996

Structural changes of tumor necrosis factor alpha associated with membrane insertion and channel formation.

Low pH enhances tumor necrosis factor alpha (TNF)-induced cytolysis of cancer cells and TNF-membrane interactions that include binding, insertion, and ion-channel formation. We have also found that TNF increases Na+ influx in cells. Here, we examined the structural features of the TNF-membrane interaction pathway that lead to channel formation. Fluorometric studies link TNF's acid-enhanced membrane interactions to rapid but reversible acquisition of hydrophobic surface properties. Intramembranous photolabeling shows that (i) protonation of TNF promotes membrane insertion, (ii) the physical state of the target bilayer affects the kinetics and efficiency of TNF insertion, and (iii) binding and insertion of TNF are two distinct events. Acidification relaxes the trimeric structure of soluble TNF so that the cryptic carboxyl termini, centrally located at the base of the trimer cone, become susceptible to carboxypeptidase Y. After membrane insertion, TNF exhibits a trimeric configuration in which the carboxyl termini are no longer exposed; however, the proximal salt-bridged Lys-11 residues as well as regional surface amino acids (Glu-23, Arg-32, and Arg-44) are notably more accessible to proteases. The sequenced cleavage products bear the membrane-restricted photoreactive probe, proof that surface-cleaved TNF has an intramembranous disposition. In summary, the trimer's structural plasticity is a major determinant of its channel-forming ability. Channel formation occurs when cracked or partially splayed trimers bind and penetrate the bilayer. Reannealing leads to a slightly relaxed trimeric structure. The directionality of bilayer penetration conforms with x-ray data showing that receptor binding to the monomer interfaces of TNF poises the tip of the trimeric cone directly above the target cell membrane.

Amino Acid Sequence↗
PubMed1996

An analysis of the dynamic range and linearity of an infrared DNA sequencer.

We are currently developing genotyping software and protocols for use on the Li-Cor model 4000(L) infrared fluorescence DNA sequencers. During the development of the genotyping software it became apparent that the potential dynamic range of the instrument was not being realized when the data collection parameters were optimized to produce a high-contrast image on the computer screen. In particular, we were unable to obtain peaks with signal to noise (S/N) greater than about 30:1 and the stronger peaks often saturated the detector. Because the numerical dynamic range available in 16-bit data collection mode exceeds 65000, our limited dynamic range of about 30 in actual data was somewhat puzzling. Hence, we undertook a study to explore the dynamic range and linearity of the Li-Cor DNA sequencer in order to minimize background fluorescence and noise as well as maximize the available S/N. Data is presented on the background and noise using different polyacrylamide gel mixes with various signal gain and offset values, and the relative contribution of the glass, gel and instrumentation to the background fluorescence is discussed. Based on these results, optimum gain and offset values were determined that maintain the background fluorescence at approximately 1-2% of the maximum dynamic range with a minimum amount of noise. Using these optimized values, we determined the detection system is linear over three orders of magnitude by titrating known quantities of an infrared fluorophore-labeled primer. In addition, we were able to detect approximately 15.2 amoles of labeled primer. The results provided by this study establish a set of guidelines for evaluating how to set the signal gain and offset in order to minimize the background and maximize the S/N and dynamic range of the Li-Cor sequencer.

DNA↗
PubMed1996

"Incisura" of the ascending aorta and vascular pedicle width in the cardiac transplant patient.

BACKGROUND: The purpose of this study was to evaluate three findings in cardiac transplant patients: the appearance, etiology, and incidence of "incisura" (a characteristic indentation) of the ascending aorta; the vascular pedicle width, which usually appears enlarged on standard chest radiographs; and clearing of the retrosternal clear space. MATERIAL: Two hundred sixty-one cardiac transplantations were performed at the University of Minnesota before December 31, 1992. The appearance, etiology, and incidence of an incisura were studied; the vascular pedicle width was measured; and the rate of clearing of the retrosternal clear space was determined. RESULTS: Forty-six percent of the patients whose ascending aorta could be visualized on the lateral chest radiogram showed an incisura. Approximately one third of the patients showed continued opacification of the retrosternal space postoperatively. The width of the vascular pedicle in this series of cardiac transplant patients measured 60.9 +/- 22.8 mm (standard deviation, 11.4 mm), in comparison to a normal of 48 +/- 5.0 mm. CONCLUSIONS: An incisura of the ascending aorta and the widened vascular pedicle are normal postoperative chest radiographic findings in cardiac transplant patients and should not be misconstrued as abnormalities.

Anastomosis, Surgical↗
PubMed1996

Complete genomic sequence and analysis of 117 kb of human DNA containing the gene BRCA1.

Over 100 distinct disease-associated mutations have been identified in the breast-ovarian cancer susceptibility gene BRCA1. Loss of the wild-type allele in > 90% of tumors from patients with inherited BRCA1 mutations indicates tumor suppressive function. The low incidence of somatic mutations suggests that BRCA1 inactivation in sporadic tumors occurs by alternative mechanisms, such as interstitial chromosomal deletion or reduced transcription. To identify possible features of the BRCA1 genomic region that may contribute to chromosomal instability as well as potential transcriptional regulatory elements, a 117,143-bp DNA sequence encompassing BRCA1 was obtained by random sequencing of four cosmids identified from a human chromosome 17 specific library. The 24 exons of BRCA1 span an 81-kb region that has an unusually high density of Alu repetitive DNA (41.5%), but relatively low density (4.8%) of other repetitive sequences. BRCA1 intron lengths range in size from 403 bp to 9.2 kb and contain the intragenic microsatellite markers D17S1323, D17S1322, and D17S855, which localize to introns 12, 19, and 20, respectively. In addition to BRCA1, the contig contains two complete genes: Rho7, a member of the rho family of GTP binding proteins, and VAT1, an abundant membrane protein of cholinergic synaptic vesicles. Partial sequences of the 1A1-3B B-box protein pseudogene and IFP 35, an interferon induced leucine zipper protein, reside within the contig. An L21 ribosomal protein pseudogene is embedded in BRCA1 intron 13. The order of genes on the chromosome is: centromere-1FP 35-VAT1-Rho7-BRCA1-1A1-3B-telomere.

Breast Neoplasms↗
PubMed1996

High-throughput cDNA screening utilizing a low order neural network filter.

A low order neural network-based filter was designed as a rapid screening agent for single-spanning transmembrane regions in an integrated informatics system. A rapid screening algorithm was seen as a compromise between costly structure-specific techniques and simple rules that gave a high false-positive rate for cDNA. The filter was applied to a library of 2123 anonymous cDNA sequences, which resulted in 61 detections. Evaluation of the detections with two other dissimilar computer prediction algorithms yielded strong transmembrane predictions for 15 of the detections, while 8 of the detections resulted in a definitive negative result. Homology searches performed on the sequences with detection reports yielded 13 homologs in the predicted reading frame, four of which are membrane associated.

Animals↗
PubMed1996

Identifying DNA polymorphisms in human TCRA/D variable genes by direct sequencing of PCR products.

The T-cell receptor (TCR) is a highly variable molecule composed of two polypeptide chains that recognize antigenic peptides in the context of major histocompatibility complex (MHC) molecules. In this study, we describe a sequence-based search for germline polymorphisms in the variable (V) gene segments of the human TCRA/D locus. Thirty different V gene segments were amplified from six to eight unrelated individuals and sequenced from low melting point agarose. Twenty-seven polymorphisms were identified in 15 V gene segments. These polymorphisms are mainly single nucleotide substitutions, but an insertion/deletion polymorphism and a single dinucleotide repeat with variable length were also seen. Of the 15 sequence variations found in the coding regions, six are silent and nine encode amino acid changes. All of the amino acid changes are found at non-conserved residues, frequently in the hypervariable regions, where they may influence MHC and/or peptide recognition. Therefore, it is possible that germline variations in TCR genes could influence an individual's immune response, and may also contribute to susceptibility to diseases such as autoimmunity.

Amino Acid Sequence↗
PubMed1995

Frequency and polymorphism of simple sequence repeats in a contiguous 685-kb DNA sequence containing the human T-cell receptor beta-chain gene complex.

The human T-cell receptor beta-chain (TCRB) gene complex spans 575 kb in chromosome region 7q35 and has been the subject of a large-scale DNA sequencing effort. A contiguous 685-kb DNA sequence from this region was searched by computer analysis for the occurrence of simple sequence repeats (microsatellites) with core sequence lengths of 2-5 nucleotides. Twenty-nine such microsatellites of repeat number n > or = 9 were found, with the majority being dinucleotide repeats. By PCR analysis, 19 were found to be polymorphic in repeat number, thus averaging one per 36 kb. These polymorphic di-, tri-, and tetranucleotide repeats had between 3 and 15 differently sized alleles each. The potential usefulness of these TCRB microsatellites for detecting disease susceptibility alleles was examined by measuring the linkage disequilibrium between these markers and flanking biallelic mutations. All but 4 microsatellites (79%) demonstrated significant linkage disequilibrium (P < 0.0001). This present study highlights the utility and potential outcomes of large-scale DNA sequencing for the identification of polymorphic simple sequence repeats.

Alleles↗