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G F Hollis

Publications and source records attributed to G F Hollis.

At least 19 recordsLinked to original sources
PubMed1997

Recombinant human 92-kDa type IV collagenase/gelatinase from baculovirus-infected insect cells: expression, purification, and characterization.

Human 92-kDa type IV collagenase/gelatinase (MMP9) has been expressed in insect cells and secreted into the cell medium via a baculovirus expression system. The expression level of the proenzyme from Trichoplusia ni cells was estimated to be > = 300 mg/L of cell medium. The recombinant protein was purified in a single step using heparin-affinity chromatography with an overall yield of ca. 70%. The purified zymogen could be activated in vitro using 4-aminophenylmercuric acetate to yield an active protease. Kinetic analysis of the activated recombinant enzyme demonstrates that this material is comparable to activated MMP9 from natural human sources. The recombinant enzyme provides a useful source of protein for a variety of biochemical and biophysical studies aimed at elucidating the structure and function of human MMP9.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of the human fertilin beta subunit.

Fertilin, a heterodimeric sperm surface protein, first identified in guinea pig, has been shown to play an important role in sperm-egg interactions. We report here the complementary DNA and deduced amino acid sequence of human fertilin beta. The human fertilin beta shares significant sequence homology with mouse, guinea pig and monkey fertilin beta and also exhibits similar structural organization. Of particular interest, the mature guinea pig fertilin beta contains an amino-terminal 90 amino acid disintegrin domain. It has been suggested that the integrin recognition sequence (TDE) of the guinea pig fertilin beta disintegrin domain mediates sperm-egg binding. The amino acid sequence at this position in human fertilin beta differs from the mouse, guinea pig and monkey sequence (mouse-QDE; human-FEE; monkey-FDE).

ADAM Proteins↗
PubMed1996

Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris.

A recombinant form of hirudin (HIR), a potent thrombin inhibitor derived from the leech Hirudo medicinalis, was cloned and expressed in the methylotrophic yeast Pichia pastoris. The HIR gene was inserted into the P. pastoris pPic9K expression vector such that the gene's expression is under alcohol oxidase (AOX1) promoter control and the HIR coding sequence is fused to the Saccharomyces cerevisiae pre-pro alpha-mating factor signal sequence. A Tn903Kan(r) determinant and His4+ gene are also present on pPic9K, affording a method for selecting chromosomal integrants of the HIR gene. Following electroporation of the DNA into the P. pastoris strain GS115 (his-4), His+ transformants were recovered and plated on medium containing increasing concentrations of the aminoglycoside antibiotic G418. The resulting His+ G418-resistant transformants were grown in shake flasks and screened for those that secreted recombinant hirudin (rHIR) to the growth medium. Clones exhibiting rHIR production and secretion were retained for fermentation studies where optimization of growth conditions was found to dramatically increase rHIR expression. One clone that was retained for further characterization secreted rHIR at a level of 1.5 g/liter. Using a straightforward two-step chromatography procedure, the rHIR was purified to > 97% with a recovery yield of 63%. The purified rHIR had the predicted N-terminal amino acid sequence and exhibited the same thrombin inhibition kinetics as a variety of HIR isoforms produced in other heterologous systems. Based on these data, P. pastoris offers an efficient system for production and purification of multigram quantities of biologically active rHIR for structure/function analyses.

Base Sequence↗
PubMed1995

Enhancing the avidity of a human recombinant anti-HIV-1 monoclonal antibody through oligomerization.

The oligomerization by chemical cross-linking of a recombinant human antiviral monoclonal antibody (MAb), r447-1, and its characterization are described. This MAb binds to an epitope residing in the hypervariable V3 region of the envelope protein (gp120/160) of HIV-1. A dimeric form of this MAb displays enhanced avidity and was found to be capable of neutralizing a greater variety of lymphoid cell culture-adapted HIV-1 variants and HIV-1 primary isolates than its monomeric form. The superior binding and breadth of reactivity of this antibody suggests it may have utility as a therapeutic and/or prophylactic agent, if it possesses an appropriate safety and immunogenicity profile.

Antibodies, Monoclonal↗
PubMed1995

Sequence of the dog immunoglobulin alpha and epsilon constant region genes.

The immunoglobulin alpha (IGHAC) and epsilon (IGHEC) germline constant region genes were isolated from a dog liver genomic DNA library. Sequence analysis indicates that the dog IGHEC gene is encoded by four exons spread out over 1.7 kilobases (kb). The IGHAC sequence encompasses 1.5 kb and includes all three constant region coding exons. The complete exon/intron sequence of these genes is described.

Amino Acid Sequence↗
PubMed1995

Use of a novel mutagenesis strategy, optimized residue substitution, to decrease the off-rate of an anti-gp120 antibody.

We have developed a novel strategy to decrease the antibody:antigen off-rate which we call optimized residue substitution. This strategy employs alanine substitution to first identify residues non-optimal for binding, as evidenced by a decrease in off-rate upon alanine replacement. These positions are then individually randomized to all amino acids, and the best replacement for each position determined. Finally, a construct which combines all optimized substitutions is generated and evaluated. We applied this strategy to the heavy chain CDR3 of P5Q, a scFv antibody which recognizes an epitope on the V3 loop of HIV gp120. We identified two amino acid substitutions that together decrease the off-rate by nearly ten-fold. The contributions by the two substitutions were near additive, indicative of independent affects on binding. We suggest that this strategy can be generalized to strengthen protein:ligand and protein:protein interactions in other systems.

Alanine↗
PubMed1994

A microplate assay for hyaluronidase and hyaluronidase inhibitors.

A sensitive, high-throughput assay has been developed which measures hyaluronidase activity in a microplate format. In this assay, hyaluronic acid is suspended in agarose in a microtiter well, the plate is incubated with the hyaluronidase solution, and the undigested hyaluronic acid is precipitated with cetylpyridinium chloride. The precipitate blocks light transmittance and therefore an increase in the visible light transmitted correlates with the amount of digested hyaluronic acid. Using this assay, as little as 0.05 units of hyaluronidase activity can be detected. The assay is highly reproducible and can be run with commercially available reagents. Hyaluronidase activity is easy to quantitate using a microplate reader and this format allows large numbers of samples to be assayed.

Chamomile↗
PubMed1993

Induction of peri-insulitis but not diabetes in islet transplants expressing a single foreign antigen. A multi-stage model of disease.

We have created a new transgenic model in which the human Epstein-Barr virus receptor, CR2 (CD21), has been expressed in pancreatic beta-cells. Mice derived from three transgenic founders bred into H-2b (C57BL/6) or H-2k (CBA) genetic backgrounds did not become spontaneously diabetic. After transplantation of CR2-expressing islets under the kidney capsule of genetically matched recipients, a histologic picture of peri-insulitis was found. However, animals did not manifest cellular invasion of the islets, destruction of the islets, or diabetes for at least 230 days. Significant numbers of both T and B lymphocytes were found in the cell population surrounding the islets. A pronounced serologic response to CR2 was also present and appeared to precede the onset of peri-insulitis. Thus, in this model, we have separated the process of diabetes induction into at least two phases. One is associated with peri-islet infiltration and an antibody response. However, at least one second signal is likely necessary for the process of islet destruction to follow.

Animals↗
PubMed1993

Characterization of cloned human endothelin receptors.

Two subtypes of human endothelin receptors, ETA and ETB, have been cloned and stably expressed in Chinese Hamster Ovary cells. These receptors have been characterized by [125I]-endothelin-1 binding and phosphatidyl inositol hydrolysis using the potent peptidyl ETA antagonists BQ-123 and BQ-153, as well as the potent ETB agonist, sarafotoxin S6c. In binding studies, Ki values for BQ-123 and BQ-153 are 17 nM and 13 nM for ETA compared to 11,100 nM and 7200 nM for ETB. Conversely, Ki values for sarafotoxin S6c are 2800 nM for ETA and 0.29 nM for ETB. Endothelin-1 stimulates phosphatidyl inositol hydrolysis in cells expressing either ETA or ETB with EC50 values of 0.2-0.3 nM, while sarafotoxin S6c stimulates phosphatidyl inositol hydrolysis only in ETB expressing cells with an EC50 value of 0.2 nM, consistent with the binding data. Comparison of binding data for the cloned and expressed human receptors with binding data for receptors obtained from human tissues indicates the cloned and expressed receptors are essentially indistinguishable from the naturally occurring receptors.

Animals↗
PubMed1991

Genomic structure of the human Ig lambda 1 gene suggests that it may be expressed as an Ig lambda 14.1-like protein or as a canonical B cell Ig lambda light chain: implications for Ig lambda gene evolution.

In pre-B cells, immunoglobulin mu (Ig mu) is associated with pre-B cell-specific proteins to form a multimeric complex that is found on the cell surface. One of these proteins is encoded by the three exon Ig lambda-like gene 14.1, whose expression is restricted to pre-B cells and occurs from an unrearranged gene. A comparison of the 14.1 gene structure to the seven-gene human Ig lambda locus revealed that the most 5' gene, Ig lambda 1, is organized in a three-exon structure very similar to the 14.1 gene. Transcription and splicing of these three-exon sequences would lead to an mRNA with an open reading frame which could encode a light (L) chain-like protein with a molecular weight of 23,045. Our analysis suggests that two transcripts may be produced from the Ig lambda 1 gene that share the same Ig lambda 1 constant region-containing third exon. One transcript would include all three 14.1-related exons and be expressed from the germline gene, and the second transcript would be produced after variable-joining (V-J) recombination has occurred to Ig lambda J1 and would encode a classic Ig lambda L chain protein. The conservation of the genomic organization of the human 14.1 and Ig lambda 1 genes and the mouse homolog, lambda 5, relative to the classic Ig lambda L chain genes provides insight into the evolution of Ig genes.

Amino Acid Sequence↗
PubMed1991

A rapid method for purifying PCR products for direct sequence analysis.

An HPLC approach for purification and sequencing of double-stranded DNA obtained directly from a PCR is described. This simple and reliable procedure has several advantages; the DNA fragment is rapidly eluted (less than 7 minutes), requires no organic cleanup, produces several hundred bases of sequence and is sensitive enough to obtain DNA sequence from a single 100-microliters PCR. This method is demonstrated by sequencing tumor necrosis factor alpha (TNF alpha) gene amplified from mouse tail DNA.

Animals↗
PubMed1989

Genomic structure of murine beta-1,4-galactosyltransferase.

We have isolated a series of overlapping murine genomic DNA clones that include the complete coding sequence of the Golgi membrane bound marker enzyme beta-1,4-galactosyltransferase. The coding sequence is distributed into six exons spanning 50,000 b.p. of mouse chromosome 4. The COOH terminal domain is predominantly encoded by exons 2-6 and the transmembrane and amino terminal cytoplasmic domains are encoded by exon 1. S1 analysis establishes the most 5' transcriptional initiation site 190 b.p. upstream of the first methionine residue.

Amino Acid Sequence↗
PubMed1989

Immunoglobulin lambda light-chain-related genes 14.1 and 16.1 are expressed in pre-B cells and may encode the human immunoglobulin omega light-chain protein.

Human pre-B cells, which produce immunoglobulin heavy chain but do not produce immunoglobulin light chain, are shown to contain a 1-kilobase transcript homologous to immunoglobulin lambda light-chain genes. Detailed analysis of RNA and cDNA clones derived from these transcripts reveals that they originate from the distinct immunoglobulin lambda-like genes 14.1/16.1. Sequence analysis of these clones reveals a long open reading frame, beginning with an ATG, capable of encoding a protein of 214 amino acids with an unprocessed molecular weight of 22,944. The C-terminal half of this predicted protein is highly homologous to immunoglobulin lambda light-chain joining and constant region protein sequence, while the amino-terminal end does not share homology with variable regions. Unlike immunoglobulin genes, these genes do not undergo rearrangement prior to expression. Analysis of a panel of 26 hematopoietic cell lines reveals that expression of 14.1/16.1 is limited to pre-B cells and one B-cell line, which, like the pre-B cells, is surface immunoglobulin negative. Antisera raised against a peptide whose sequence was predicted from the 14.1 cDNA sequence identifies a 22-kDa protein in human pre-B cells. Immunoprecipitation of immunoglobulin mu-chain from these pre-B cells with anti-immunoglobulin mu antibody coprecipitates a 22-kDa protein, which is a candidate for the human immunoglobulin omega light-chain protein and may be the protein product of the 14.1/16.1 genes.

Amino Acid Sequence↗
PubMed1988

Characterization of the full length cDNA for murine beta-1,4-galactosyltransferase. Novel features at the 5'-end predict two translational start sites at two in-frame AUGs.

We have isolated overlapping cDNA clones representing the full length (4038 base pairs) transcript for murine beta-1,4-galactosyltransferase. The coding sequence predicts a membrane-bound glycoprotein with three distinct structural features, a large COOH-terminal domain (355 amino acids), a single transmembrane domain (20 amino acids), and a short NH2-terminal domain. Primer extension analysis, S1 protection analysis, and RNA blotting demonstrate the presence of two sets of mRNA transcripts which differ in length by about 200 base pairs. The 5' boundary of the long transcripts maps upstream of two in-frame ATGs. The 5' boundary of the short transcripts maps between these two ATGs. These results predict that two related forms of beta-1,4-galactosyltransferase of 399 and 386 amino acids are synthesized as a consequence of alternative translation initiation. The long form (399 amino acids) has an NH2-terminal extension of 13 amino acids.

Amino Acid Sequence↗
PubMed1988

Detection of genes of interest in tissues and cells by in situ hybridization.

In situ hybridization (ISH) is a powerful technique for assessing the expression of particular genes of interest within individual cells in tissues or cytospins. The only limitations of the technique are the availability of appropriate probes and tissues. When coupled with routine histology and DNA analysis, characterization and classification of tissues can be greatly extended. Future applications include the identification of tumor-specific markers that can be combined with ISH in diagnostic strategies.

Animals↗
PubMed1988

Evidence for two forms of murine beta-1,4-galactosyltransferase based on cloning studies.

We have isolated overlapping cDNA clones representing the full-length transcript (4038 base pairs) for murine beta-1,4-galactosyltransferase. The coding sequence predicts a membrane-bound glycoprotein with 3 distinct structural features: 1) a large, potentially glycosylated COOH-terminal domain (355 amino acids) which is positioned within the Golgi lumen and contains both the catalytic and alpha-lactalbumin binding site; 2) a single transmembrane domain (20 amino acids); and 3) a short NH2-terminal domain containing 2 Met residues, separated by 12 amino acids. The gene for murine beta-1,4-galactosyltransferase is unusual in that it specifies 2 mRNA transcripts which differ in length by about 200 base pairs. The longer transcript contains both Met residues found in the NH2-terminal domain; the shorter transcript contains only the downstream Met. These results predict that 2 related forms of beta-1,4-galactosyltransferase of 399 and 386 amino acids are synthesized as a consequence of alternative translation initiation. Both forms of the enzyme are identical in primary structure with the exception that the long form has an NH2-terminal extension of 13 amino acids which, in part, potentially encodes a cleavable signal sequence. The structural implications, topological distribution and potential biological significance of the 2 forms of the enzyme are discussed.

Amino Acid Sequence↗
PubMed1988

Complex translocation disrupts c-myc regulation in a human plasma cell myeloma.

A complex translocation has interrupted the third exon of the c-myc gene in human plasma cell myeloma tumor cells and a derivative cell line (NCI-H929). As a result of this rearrangement, a chimeric mRNA is expressed which commences 5' of the c-myc coding region and includes sequences introduced by the translocation event. All of the detectable c-myc-containing mRNA in the tumor and cell line was derived from this rearranged c-myc allele. This chimeric c-myc mRNA, in which most of the germ line c-myc 3' untranslated region has been replaced, was greater than sevenfold more stable than c-myc transcripts with intact 3' ends. This suggests that the 3' untranslated region may play an important role in c-myc mRNA stability.

Base Sequence↗