Protein sequence analysis: automated microsequencing.

The automated microsequencing of proteins can now be carried out at the 5- to 10-picomoles (submicrogram) level on polypeptides obtained directly from one- and two-dimensional gel electrophoresis. The techniques are applicable to polypeptides ranging in size from small peptides (less than 10 residues) to large proteins (more than 1000 residues).

Human platelet-derived growth factor (PDGF): amino-terminal amino acid sequence.

Human platelet-derived growth factor (PDGF) obtained from outdated human platelets was subjected to amino-terminal amino acid sequence analysis by automated Edman degradation. Despite the apparent presence of limited proteolytic degradation of the protein derived from this method, the sequence analysis reveals two primary peptide sequences and suggests that active PDGF is composed of two, possibly homologous, peptides linked by a disulfide bond or bonds.

Contemporary methodology for protein structure determination.

The techniques used for the characterization of protein and peptide structure have undergone great changes that have improved the speed, reliability, and applicability of the process. High-performance liquid chromatography and gel electrophoresis have made the purification of proteins and peptides a routine procedure, even when the compound of interest is a minor component of a complex biological mixture. The chemistry and instrumentation used in amino acid analysis and amino acid sequencing now permit the analysis of as little as 5 to 50 picomoles of samples. This represents an increase in sensitivity of more than a thousandfold over the last 10 years and has made possible the structural analysis of a wide variety of scarce but important compounds.

Rat transforming growth factor type 1: structure and relation to epidermal growth factor.

The complete amino acid sequence of rat transforming growth factor type 1 has been determined. This growth factor, obtained from retrovirus-transformed fibroblasts, is structurally and functionally related to mouse epidermal growth factor and human urogastrone. Production of this polypeptide by various neoplastic cells might contribute to the continued expression of the transformed phenotype.

Mammalian muscle acetylcholine receptor: a supramolecular structure formed by four related proteins.

The nicotinic acetylcholine receptor has been purified from fetal calf muscle. Amino terminal amino acid sequence data indicate that the mammalian receptor is formed from closely related but distinct subunits. A cytoskeletal component, actin, may be associated with the receptor.

Human fibroblast interferon: amino acid analysis and amino terminal amino acid sequence.

The purification of human fibroblast interferon has been simplified to a two-step procedure consisting of affinity chromatography on Blue Sepharose and sodium dodecyl sulfate polyacrlamide gel electrophoresis. A preliminary amino acid composition and the sequence of the 13 amino-terminal residues of homogeneous interferon prepared by this method is reported.

Acetylcholine receptor: complex of homologous subunits.

The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

Amino terminal sequence of the major component of human lymphoblastoid interferon.

Homogeneous human lymphoblastoid interferon with an apparent molecular size of 18,500 daltons was characterized by its amino acid composition. Analysis of the amino terminal sequence by Edman degradation indicates that the sequence is unique.

Mouse interferons: amino terminal amino acid sequences of various species.

Mouse interferons of three size classes (A, 35,000 to 40,000 daltons; B, 26,000 to 33,000 daltons; and C, 20,000 daltons) were purified from Ehrlich ascites tumor cells infected with Newcastle disease virus. The sequences of the first 24 amino acids (No. 17 has not been identified) of interferons A and B are identical. The sequence of the first 20 amino acids of interferon C differs from that of A and B in 18 positions. There is partial homology in amino terminal sequence between mouse interferons A (or B) and a human fibroblast interferon and between mouse interferon C and a human lymphoblastoid interferon.

Simian sarcoma virus onc gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor.

The transforming protein of a primate sarcoma virus and a platelet-derived growth factor are derived from the same or closely related cellular genes. This conclusion is based on the demonstration of extensive sequence similarity between the transforming protein derived from the simian sarcoma virus onc gene, v-sis, and a human platelet-derived growth factor. The mechanism by which v-sis transforms cells could involve the constitutive expression of a protein with functions similar or identical to those of a factor active transiently during normal cell growth.

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.

Advances in DNA sequencing technology.

Efforts to map and sequence the genomes of the human and other species have stimulated efforts to improve the technology required for these endeavors. During the last year, these efforts have produced substantial advances in DNA template preparation, sequencing chemistry, and gel analysis.

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.

Bovine luteinizing hormone iodinated at alpha Tyr21, alpha Tyr92, or alpha Tyr93 retains specific binding activity.

It has previously been shown that as the extent of iodination or nitration of LH is increased, receptor-binding activity is lost. To determine whether this loss is attributable to modification of a specific tyrosine, we located iodotyrosines in only those iodinated molecules that retained specific binding activity. Iodinated bovine LH (*bLH) with intact binding activity was separated from *bLH lacking activity by binding to and elution from receptors. Gel exclusion chromatography of tryptic peptides and microsequenator analysis of tryptic glycopeptides showed that iodotyrosine was present at each of the only readily accessible residues in intact hormone: alpha Tyr21, alpha Tyr92, and alpha Tyr93. Loss of activity with increased modification could not be explained by subunit dissociation, hormone aggregation, or degradative release of radioactive residues. These results together with the previous finding that those molecules of *bLH that can bind specifically to receptors do so with an apparent Ka indistinguishable from that of unmodified hormone show that any one of the residues, alpha Tyr21, alpha Tyr92, or alpha Tyr93, can be iodinated without an effect on binding and suggest that none of these residues interacts directly with receptor. They further suggest that it is modification of more than one tyrosine in the same molecule which negatively affects binding. We discuss how modification of two tyrosines might decrease binding activity when modification of any one has no observable effect.

Purification and characterization of canine intestinal motilin.

A 22 amino acid peptide with motilin-like immunoreactivity was purified from acetic acid extracts of small intestinal mucosa from mongrel dogs. Sequential chromatography on carboxymethyl-cellulose, Sephadex G-50, CM cellulose, Biogel P6, and two steps of high-performance liquid chromatography were used for purification. Microsequence analysis of the purified product permitted unambiguous identification of residues 2-22 as -VPIFTHSELQKIREKERNKGQ. The sequence of porcine intestinal motilin is FVPIFTYGELQRMQEKERNKGQ. The amino terminal residue of the canine peptide could not be assigned with certainty since Phe, Lys, and Ser all were identified by analysis of PTH derivatives on the first sequencing cycle. Definite amino acid differences between canine and porcine motilin thus were identified in positions 7, 8, 12, 13 and 14. These differences did not alter immunoreactivity of canine motilin with antibodies specific for the carboxyl-terminal portion of porcine motilin, but probably explain markedly diminished immunoreactivity with antibodies to the amino or mid-portion of porcine motilin. Synthetic Phe1 canine motilin was prepared by a solid phase method. The synthetic peptide had the same pattern of immunoreactivity as natural canine motilin and was biologically active with a potency similar to synthetic porcine motilin for induction of premature activity fronts of the interdigestive motor complex in the small intestine of fasting dogs.