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M Hunkapiller

Publications and source records attributed to M Hunkapiller.

15 recordsLinked to original sources
PubMed2001

The sequence of the human genome.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

Algorithms↗
PubMed1984

Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography.

Bovine bone morphogenetic protein (bBMP) induces differentiation of mesenchymal-type cells into cartilage and bone. bBMP has an apparent Mr of 18,500 +/- 500 and represents less than 0.001% of the wet weight of bone tissue. A Mr 34,000 protein resembling osteonectin is separated by extraction with Triton X-100. A Mr 24,000 protein and about half of a Mr 22,000 protein are disassociated from bBMP by precipitation in 1.5 M guanidine hydrochloride. Aggregates of bBMP and a Mr 14,000 protein are insoluble in aqueous media; the bBMP becomes soluble when the Mr 14,000 protein is disassociated in 6 M urea and removed from the solution by ultrafiltration. Three separate molecular species with apparent Mrs 18,500, 17,500, and 17,000 are eluted at 0.10, 0.15, and 0.20 M phosphate ion concentrations, respectively, from a hydroxy-apatite column. The Mr 18,500 protein has the amino acid composition of acidic polylpeptide and includes four half-cystine residues; the pI is 4.9-5.1. The Mr 22,000 component is a chromoprotein resembling ferritin. The NH2-terminal amino acid sequence of the Mr 17,500 protein simulates histone H2B. The Mr 17,000 protein may possess calmodulin activity. Aggregates of the Mr 18,500 and other proteins induce formation of large deposits of bone; the Mr 18,500 protein alone is rapidly absorbed and induces formation of small deposits. None of the other proteins induces bone formation.

Amino Acid Sequence↗
PubMed1984

Biologic activity in a fragment of recombinant human interferon alpha.

To attempt to locate functionally important regions of the interferon (IFN) molecule, recombinant human IFN-alpha 2 was subjected to proteolytic digestion. The bacterial proteinase thermolysin produced two major complementary fragments, HuIFN-alpha 2-(1-110) and HuIFN-alpha 2-(111-153). After reduction with 2-mercaptoethanol and separation of the two major fragments on NaDodSO4/polyacrylamide gel electrophoresis, antiviral activity persisted in the larger, Mr 12,000, fragment consisting of the amino-terminal 110 amino acids.

Animals↗
PubMed1983

Major intrinsic polypeptide of lens membrane. Biochemical and immunological characterization of the major cyanogen bromide fragment.

A protein of Mr 26000 has been shown to be the major component of eye-lens junctions, which are similar but not identical to the gap junctions of liver and other tissues. Cyanogen bromide cleavage of the Mr 26000 polypeptide from bovine lenses yields a major fragment of Mr 15000 (fragment 1). However, if the junctions are first treated with trypsin or carboxypeptidase Y, cyanogen bromide treatment yields a fragment of reduced molecular weight. Since protease treatment has been shown to cleave residues almost exclusively from the carboxy-terminal end of the Mr 26000 polypeptide, it follows that fragment 1 represents the carboxy-terminal half of this molecule, part of which is exposed to proteolytic attack outside the membrane. This latter result is corroborated by the fact that antisera which recognize both the Mr 26000 polypeptide and fragment 1 fail to do so after preadsorption with intact membranes. In addition, comparative amino acid and partial sequence analyses of the Mr 26000 polypeptide and fragment 1 indicate that fragment 1 is more hydrophilic in character, suggesting that much of the amino-terminal half of the Mr 26000 polypeptide is buried within the lipid bilayer.

Amino Acid Sequence↗
PubMed1982

Cuticle protein genes of Drosophila: structure, organization and evolution of four clustered genes.

Most of a 9 kb region of the Drosophila genome containing genes for several cuticle proteins has been sequenced. Five cuticle-gene-like sequences have been identified and mapped. Amino acid sequences of four of the five major third instar cuticle proteins have been determined. These four sequences are identical with those predicted from the sequences of four of the five genes. Two cuticle genes are transcribed in one direction and two in the opposite direction. The fifth cuticle-like gene is judged to be a pseudogene because several features of its structure and the absence of detectable transcripts suggest it is nonfunctional. Sequence comparisons indicate that it arose by an unequal crossing-over event involving two closely related and adjacent cuticle genes. Each of the four cuticle genes contains a signal peptide coding sequence interrupted by a short intervening sequence (about 60 bp) at a conserved site. Conserved sequences occur in the 5' mRNA untranslated region, in the adjacent 35 bp of upstream flanking sequence and at -200 bp from the mRNA start position in each of the cuticle genes. We discuss the structure, organization and evolution of these cuticle genes as a model small gene family.

Amino Acid Sequence↗
PubMed1982

A transposable element that splits the promoter region inactivates a Drosophila cuticle protein gene.

Two mutations that affect larval cuticle protein gene expression in the 2/3 variant Drosophila melanogaster strain were investigated. We demonstrate that this strain synthesizes an electrophoretic variant, fast 2 (CPf2), of wild-type cuticle protein 2(CP2). It also lacks detectable amounts of cuticle protein 3 (CP3). The other major cuticle proteins are still present. Protein and DNA sequence analyses indicate that point mutations cause two amino acid substitutions that change the electrophoretic mobility of CPf2 relative to that of CP2. The mutation abolishing the expression of CP3 was found to be a 7.3-kilobase DNA insertion located within the T-A-T-A box region of this gene, at -31 base pairs from the mRNA start site. This DNA insertion, called H.M.S. Beagle, belongs to a conserved family of repeated DNA elements that have characteristics similar to those of previously characterized Drosophila transposable elements. H.M.S. Beagle elements are repeated approximately 50 times in the haploid genome and exhibit restriction fragment-length polymorphisms around points of insertion between Canton S, Oregon R, and 2/3 Drosophila strains. Sequence analysis indicates that H.M.S. Beagle contains 266-base-pair direct repeats at its termini and is flanked by a duplication of 4 base pairs of target DNA sequence, T-A-T-A, in the CP3 gene insertion. Thus, insertion of a transposable element into the putative promoter region of the CP3 gene is evidently responsible for inactivating CP3 gene expression.

Animals↗
PubMed1981

Microchemical instrumentation.

A number of cell surface molecules of great theoretical and practical importance simply cannot be obtained in amounts sufficient for molecular analysis using conventional methods and instrumentation. Because of our interest in such studies, we began about eight years ago to explore the possibility of developing new instrumentation for the sequence analysis of very small quantities of polypeptide chains. These efforts have led to the development of two microsequenators which employ one thousandth to one ten-thousandth the material used in the original sequenator described by Per Edman. In addition, in conjunction with the explosion of recombinant DNA techniques, we also have begun to develop instrumentation for the sequence analysis and synthesis of DNA molecules. In this paper we describe briefly several new instruments that have been developed at Caltech. We believe this new instrumentation in conjunction with the recombinant DNA and hybridoma technologies will provide unique opportunities to analyze cell-surface molecule in the years ahead.

Amino Acid Sequence↗
PubMed1981

Porcine pituitary dynorphin: complete amino acid sequence of the biologically active heptadecapeptide.

The full primary structure of the very potent opioid peptide dynorphin, from porcine pituitary, has been determined. It is (H)Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln(OH). The synthetic peptide with this sequence behaves identically to natural dynorphin in a number of ways, and it has the same potency in the guinea pig ileum myenteric plexus--longitudinal muscle bioassay. The potency is accounted for by the first 13 residues.

Amino Acid Sequence↗
PubMed1980

Human fibroblastoid interferon: immunosorbent column chromatography and N-terminal amino acid sequence.

Three mice and one rabbit were inoculated with purified human fibroblastoid interferon. Neutralizing activity to human fibroblastoid interferon was observed in the serum of these animals with the rabbit showing the highest anti-interferon titers (10(6) neutralizing units/mL). Rabbit antiserum was coupled to cyanogen bromide activated Sepharose, and the resulting material was tested for use in the purification of human fibroblastoid interferon. Pure interferon obtained by this procedure was analyzed, and we report the sequence of the first 13 N-terminal amino acid residues of this protein.

Amino Acid Sequence↗
PubMed1980

N-Terminal sequence of human big gastrin: sequence, synthetic and immunochemical studies.

The previously assigned structure of human big gastrin is revised as a result of sequencing and immunological studies on synthetic peptides. A nonadecapeptide has been synthesized and found to have full immunochemical potency compared with natural human G34 in a radioimmunoassay which is specific for the N-terminal sequence. Syntheses of the peptides were achieved using the stepwise procedure with benzyloxycarbonyl-amino acids and fragment couplings mediated mainly by the dicyclohexylcarbodiimide procedure in the presence of either N-hydroxysuccinimide or 1-hydroxybenzotriazole. Purification of the peptide fragments was by Sephadex LH-20 chromatography and removal of protecting groups was effected using 90% trifluoroacetic acid in the presence of scavengers. Purification of the nonadecapeptide was achieved by high performance liquid chromatography.

Amino Acid Sequence↗
PubMed1979

Dynorphin-(1-13), an extraordinarily potent opioid peptide.

We describe the opioid properties of a tridecapeptide, the sequence of which corresponds to the NH2-terminal sequence of dynorphin, a novel porcine pituitary endorphin. It contains [Leu]enkephalin. In the guinea pig ileum longitudinal muscle preparation it is about 700 times more potent than [Leu]enkephalin. Its effects in this tissue are blocked completely by naloxone, but the apparent affinity of naloxone is 1/13th that for blockade of [Leu]enkephalin or normorphine. In the mouse vas deferens, this peptide is 3 times more potent than [Leu]enkephalin. Well-washed rat brain membranes degrade the peptide rapidly, suggesting the presence of a membrane-bound degradative enzyme. The peptide displays considerable immunoreactivity in assays with antisera that have been used for the immunohistochemical localization of [Leu]enkephalin. The remarkable enhancement of the potency of [Leu]enkephalin by the COOH-terminal extension -Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-OH suggests new interpretations concerning the structure of opiate receptors and the function of the enkephalin pentapeptides.

Amino Acid Sequence↗
PubMedDate not supplied

A microchemical facility for the analysis and synthesis of genes and proteins.

A series of automated instruments that use state-of-the-art chemical methods has been developed for high-sensitivity protein sequencing, DNA synthesis and peptide synthesis. These instruments have been integrated into a centralized microchemical facility in order to promote their use for the study of a variety of biologically interesting problems. This facility has as one of its major functions the development of new chemistries and instrumentation for the structural analysis and synthesis of genes and proteins.

Amino Acid Sequence↗