Biomedical subjects

W S Lane

Publications and source records attributed to W S Lane.

At least 127 records · Page 7Linked to original sources

PubMed1993

Distinct functions of SR proteins in alternative pre-mRNA splicing.

Alternative splicing of precursor messenger RNAs (pre-mRNAs) is a common mechanism of regulating gene expression. SR proteins are a family of pre-mRNA splicing factors that are structurally related and evolutionarily conserved. Any member of the SR family can complement a splicing-deficient extract that lacks the entire family of SR proteins. Here it is demonstrated that particular SR proteins have distinct functions in alternative pre-mRNA splicing in vitro. In addition, SR proteins are differentially expressed in a variety of tissues. These results suggest a fundamental role for SR proteins in the regulation of alternative splicing.

Alternative Splicing↗

PubMed1993

ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family.

Motor neurons stimulate their postsynaptic muscle targets to synthesize neurotransmitter receptors. Polypeptide signaling molecules may mediate this inductive interaction. Here we report the purification of ARIA, a protein that stimulates the synthesis of muscle acetylcholine receptors, and the isolation of ARIA cDNA. Recombinant ARIA increases acetylcholine receptor synthesis greater than 3-fold, and it induces tyrosine phosphorylation of a 185 kd muscle protein. The ARIA cDNA hybridizes with mRNAs that are expressed in the spinal cord from E4, a time prior to the onset of neuromuscular synapse formation, through adulthood. By E7, hybridizing mRNAs are concentrated in motor neurons. Chicken ARIA is homologous to the rat Neu differentiation factor and human here-gulin, ligands for the receptor tyrosine kinase encoded by the neu (c-erbB2, HER2) proto-oncogene. Our data suggest that members of the ARIA protein family promote the formation and maintenance of chemical synapses and, furthermore, that receptor tyrosine kinases play important roles in this process.

Animals↗

PubMed1993

The major fat-globule membrane proteins, bovine components 15/16 and guinea-pig GP 55, are homologous to MGF-E8, a murine glycoprotein containing epidermal growth factor-like and factor V/VIII-like sequences.

The glycoproteins, Components 15/16 and GP 55, were partially purified from bovine and guinea-pig fat-globule membrane (FGM), respectively. Peptides prepared from immunoblots of Component 16 and GP 55, were shown by sequencing, to be similar to mouse MFG-E8 and a human milk protein of M(r) 46,000. Both of these latter proteins are associated with the FGM and contain epidermal growth factor-like domains and/or factor V/VIII-like sequences. These data complement earlier biochemical studies on the biosynthesis, tissue distribution and topology of Components 15/16 and GP 55, and indicate that MFG-E8-like proteins are expressed on the apical surfaces of mammary epithelial cells and are secreted both in a soluble form and as major components of the FGM in diverse species. The possible function of these proteins in lactation and in mammary and neonatal development is discussed.

Amino Acid Sequence↗

PubMed1993

Yeast RNA polymerase II subunit RPB11 is related to a subunit shared by RNA polymerase I and III.

The characterization of RNA polymerase subunit genes has revealed that some subunits are shared by the three nuclear enzymes, some are homologous, and some are unique to RNA polymerases I, II, or III. We report here the isolation and characterization of the yeast RNA polymerase II subunit RPB11, which is encoded by a single copy RPB11 gene located directly upstream of the topoisomerase I gene, TOPI, on chromosome XV. The sequence of the gene predicts an RPB11 subunit of 120 amino acids (13,600 daltons), only two amino acids shorter than the RPB9 polypeptide, that co-migrates with RPB11 under most SDS-PAGE conditions, RPB11 was found to be an essential gene that encodes a protein closely related to an essential subunit shared by RNA polymerases I and III, AC19. RPB11 contains a 19 amino acid segment found in three other yeast RNA polymerase subunits and the bacterial RNA polymerase subunit alpha. Some mutations that affect RNA polymerase assembly map within this segment, suggesting that this region may play a role in subunit interactions. As the isolation of RPB11 completes the isolation of known yeast RNA polymerase II subunit genes, we briefly summarize the salient features of these twelve genes and the polypeptides that they encode.

Amino Acid Sequence↗

PubMed1992

Different length peptides bind to HLA-Aw68 similarly at their ends but bulge out in the middle.

We report here the determination and refinement to 1.9 A resolution by X-ray cryo-crystallography the structure of HLA-Aw68. The averaged image from the collection of bound, endogenous peptides clearly shows the atomic structure at the first three and last two amino acids in the peptides but no connected electron density in between. This suggests that bound peptides, held at both ends, take alternative pathways and could be of different lengths by bulging out in the middle. Peptides eluted from HLA-Aw68 include peptides of 9, 10 and 11 amino acids, a direct indication of the length heterogeneity of tightly bound peptides. Peptide sequencing shows relatively conserved 'anchor' residues at position 2 and the carboxy-terminal residue. Conserved binding sites for the peptide N and C termini at the ends of the class I major histocompatibility complex binding groove are apparently dominant in producing the long half-lives of peptide binding and the peptide-dependent stabilization of the class I molecule's structure.

Amino Acid Sequence↗

PubMed1992

Isolation of a large aggregating proteoglycan from human brain.

A large proteoglycan (365 kDa), identified with monoclonal antibodies raised against chondroitin sulfate, was isolated from human brain. The isolation required anion-exchange chromatography followed by gel filtration through a Sephacryl S-500 column. The proteoglycan bound specifically to [3H]hyaluronate (HA). The binding was not reduced by high salt concentrations (up to 4 M) and was inhibited at low pH (< 4.0). The binding was inhibited by the octamer and decamer (but not the hexamer) oligosaccharides of HA. Limited proteolysis of the proteoglycan gave rise to a relatively stable polypeptide (80 kDa). The amino-terminal sequence of the 80-kDa polypeptide was identical to the cDNA-derived amino-terminal sequence of versican, a large human fibroblast proteoglycan. A monoclonal antibody raised against bovine proteoglycans and recognizing the versican core protein reacted by immunoblotting with the proteoglycan isolated from human brain. The antibody was used to localize the proteoglycan in acetone-fixed cryostat sections of bovine spinal cord. The localization of the proteoglycan in the central nervous system was identical to that previously reported for glial hyaluronate-binding protein (GHAP), a 60-kDa glycoprotein of the brain extracellular matrix (ECM). However, a major difference was observed with respect to the sensitivity of the two antigens to hyaluronidase. As previously reported, GHAP was released from the tissue by hyaluronidase digestion, whereas the proteoglycan persisted under these conditions. We conclude that the protein-hyaluronate aggregates in brain ECM contain both GHAP and versican, that GHAP is only retained in the ECM by its interaction with hyaluronate, and that the proteoglycan is anchored in some other manner and probably connects cell surfaces with the ECM since it was not released by hyaluronidase digestion.

Amino Acid Sequence↗

PubMed1992

Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size.

Peptides bound to class I molecules are 8-10 amino acids long, and possess a binding motif representative of peptides that bind to a given class I allele. In the only published study of naturally processed peptides bound to class II molecules (mouse I-Ab and I-Eb), these peptides were longer (13-17 amino acids) and had heterogenous carboxy terminals but precise amino-terminal truncations. Here we report the characterization of acid-eluted peptides bound to HLA-DR1 by high-performance liquid chromatography, mass spectrometry and microsequencing analyses. The relative molecular masses of the peptides varied between 1,602 and 2,996 (13-25 residues), the most abundant individual M(r) values being between 1,700 and 1,800, corresponding to an average peptide length of 15 residues. Complete sequence data were obtained for twenty peptides derived from five epitopes, of which all but one were from self proteins. These peptides represented sets nested at both the N- and C-terminal ends. Binding experiments confirmed that all of the isolated peptides had high affinity for the groove of DR1. Alignment of the peptides bound to HLA-DR1 and the sequences of 35 known HLA-DR1-binding peptides revealed a putative motif. Although peptides bound to class II molecules may have some related features (due to the nonpolymorphic HLA-DR alpha-chain), accounting for degenerate binding to different alleles, particular amino acids in the HLA-DR beta-chains presumably define allelic specificity of peptide binding.

Amino Acid Sequence↗

PubMed1992

Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription.

We have discovered a protein termed Dr1 that interacts with the TATA-binding protein, TBP. The association of Dr1 with TBP results in repression of both basal and activated levels of transcription. The interaction of Dr1 with TBP precludes the formation of a transcription-competent complex by inhibiting the association of TFIIA and/or TFIIB with TBP. Dr1 activity is associated with a 19 kd protein. A cDNA clone encoding Dr1 was isolated. Dr1 is phosphorylated in vivo and phosphorylation of Dr1 affected its interaction with TBP. Our results suggest a regulatory role for Dr1 in repression of transcription mediated via phosphorylation.

Amino Acid Sequence↗

PubMed1992

A rapamycin-selective 25-kDa immunophilin.

FKBP25, a previously uncharacterized 25-kDa FK506- and rapamycin-binding protein, was purified to homogeneity from calf thymus, brain, and spleen, and the sequence of a 215 amino acid (aa) 24-kDa C-terminal peptide was established. The N-terminal domain (101 aa) is unrelated to any known protein, is hydrophilic, and is predicted by circular dichroism spectroscopy to be largely alpha-helix. The C-terminal domain (114 aa) is homologous to FKBP12 and other FKBPs but has a potential nuclear targeting sequence and a unique insertion of seven amino acids in one of its loops. FKBP25 displays the rotamase activity characteristic of FKBPs; the activity is inhibited by the immunosuppressants rapamycin (Ki = 0.9 nM) and FK506 (Ki = 160 nM), but not cyclosporin A. The protein, its rapamycin selectivity, and the potential nuclear targeting sequence are discussed in terms of the structure of hFKBP12.

Amino Acid Sequence↗

PubMed1992

Transmission-blocking antibodies recognize microfilarial chitinase in brugian lymphatic filariasis.

Brugia malayi is a parasitic nematode that causes lymphatic filariasis in humans. The monoclonal antibody MF1, which mediates clearance of peripheral microfilaremia in a gerbil infection model, recognizes two stage-specific proteins, p70 and p75, in B. malayi microfilariae. cDNA coding for the MF1 antigen was sequenced, and the predicted protein sequence shows significant similarities to chitinases from bacteria and yeast. When microfilarial extracts and purified preparations of the MF1 antigen were tested for chitinase activity, strong bands of chitin-degrading activity comigrated in SDS/PAGE with p70 and p75 and showed a reduction-dependent mobility shift characteristic of the MF1 antigen. Thus, the MF1 antigen is microfilarial chitinase, which may function to degrade chitin-containing structures in the microfilaria or in its mosquito vector during parasite development and transmission.

Amino Acid Sequence↗

PubMed1992

Isolation of two genes that encode subunits of the yeast transcription factor IIA.

The yeast transcription factor IIA (TFIIA), a component of the basal transcription machinery of RNA polymerase II and implicated in vitro in regulation of basal transcription, is composed of two subunits of 32 and 13.5 kilodaltons. The genes that encode these subunits, termed TOA1 and TOA2, respectively, were cloned. Neither gene shares obvious sequence similarity with the other or with any other previously identified genes. The recombinant factor bound to a TATA binding protein-DNA complex and complemented yeast and mammalian in vitro transcription systems depleted of TFIIA. Both the TOA1 and TOA2 genes are essential for growth of yeast.

Amino Acid Sequence↗

PubMed1992

Identification of novel members of the serum amyloid A protein superfamily as constitutive apolipoproteins of high density lipoprotein.

A novel serum amyloid A protein (SAA) has been identified as a normal apolipoprotein component of non-acute phase high density lipoprotein. This novel SAA has been designated "constitutive" SAA (C-SAA) to distinguish it from "acute phase" SAA (A-SAA). C-SAA was partially sequenced, and immunochemical analyses indicated that it constitutes a distinct subclass of apolipoproteins within the SAA superfamily. A C-SAA cDNA clone was isolated from a human liver library and sequenced. The clone predicts a pre-C-SAA molecule of 130 residues from which an 18-residue leader peptide is cleaved. The 112-residue mature molecule is 8 residues longer than human A-SAA; the size difference is due to the presence of an octapeptide between positions 70 and 77 that is not found in the corresponding region of human A-SAA. Paradoxically, octapeptides of similar composition are found at similar positions in the A-SAAs of a number of other species. The C-SAA octapeptide specifies the first two residues of a NSS tripeptide, the only potential N-linked glycosylation site in the molecule. Studies indicate that approximately 50% of these sites are glycosylated, thereby giving rise to two size classes, 14 and 19 kDa, of C-SAA in vivo. Human acute phase liver contains little C-SAA mRNA relative to the levels of A-SAA mRNA, and the treatment of PLC/PRF/5 hepatoma cells with monocyte-conditioned medium does not induce C-SAA mRNA concentrations to detectable levels, in contrast to the massive induction of A-SAA mRNA observed. C-SAA is therefore not a major acute phase reactant.

Amino Acid Sequence↗

PubMed1992

SR proteins: a conserved family of pre-mRNA splicing factors.

We demonstrate that four different proteins from calf thymus are able to restore splicing in the same splicing-deficient extract using several different pre-mRNA substrates. These proteins are members of a conserved family of proteins recognized by a monoclonal antibody that binds to active sites of RNA polymerase II transcription. We purified this family of nuclear phosphoproteins to apparent homogeneity by two salt precipitations. The family, called SR proteins for their serine- and arginine-rich carboxy-terminal domains, consists of at least five different proteins with molecular masses of 20, 30, 40, 55, and 75 kD. Microsequencing revealed that they are related but not identical. In four of the family members a repeated protein sequence that encompasses an RNA recognition motif was observed. We discuss the potential role of this highly conserved, functionally related set of proteins in pre-mRNA splicing.

Amino Acid Sequence↗

PubMed1992

The third subunit of protein phosphatase 2A (PP2A), a 55-kilodalton protein which is apparently substituted for by T antigens in complexes with the 36- and 63-kilodalton PP2A subunits, bears little resemblance to T antigens.

The small and middle T (tumor) antigens of polyomavirus have been shown previously to associate with the 36-kDa catalytic subunit and the 63-kDa regulatory subunit of protein phosphatase type 2A, apparently substituting for a normal third 55-kDa regulatory subunit (D.C. Pallas, L.K. Shahrik, B.L. Martin, S. Jaspers, T.B. Miller, D.L. Brautigan, and T.M. Roberts, Cell 60:167-176, 1990). To facilitate a comparison of the normal regulatory subunit and T antigens, we isolated a 2.14-kb cDNA clone encoding this 55-kDa subunit from a rat liver library. Using a probe from the coding region of this gene, we detected a major 2.4-kb mRNA transcript in liver and muscle RNAs. The 55-kDa protein phosphatase 2A subunit purified from rat skeletal muscle generates multiple species when analyzed on two-dimensional gels. Transcription and translation of the clone in vitro produced a full-length protein that comigrated precisely on two-dimensional gels with three of these species, indicating that the 55-kDa protein is apparently modified similarly in vivo and in reticulocyte lysates. Additional species in the purified preparation were not found in the translate, suggesting that there are probably two or more isoforms of this protein in rat muscle. Somewhat surprisingly, there was no clear homology with T-antigen amino acid sequences.

Amino Acid Sequence↗

PubMed1991

Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase.

The overproduction, purification, and determination of the active-site catalytic nucleophile of the DNA (cytosine-5)-methyltransferase (DCMtase) enzyme M.HaeIII are reported. Incubation of purified M.HaeIII with an oligodeoxynucleotide specifically modified with the mechanism-based inhibitor 5-fluoro-2'-deoxycytidine [Osterman, D. G., et al. (1988) Biochemistry 27, 5204-5210], in the presence of the cofactor S-adenosyl-L-methionine (AdoMet), resulted in the formation of a covalent DNA-M.HaeIII complex, which was purified to homogeneity. Characterization of the intact complex showed it to consist of one molecule of the FdC-containing duplex oligonucleotide, one molecule of M.HaeIII, and one methyl group derived from AdoMet. Exhaustive proteolysis, reduction, and alkylation of the DNA-M.HaeIII complex led to the isolation of two DNA-bound peptides--one each from treatment with Pronase or trypsin--which were subjected to peptide sequencing in order to identify the DNA attachment site. Both peptides were derived from the region of M.HaeIII containing a Pro-Cys sequence that is conserved in all known DCMtases. At the position of this conserved Cys residue (Cys71), in the sequence of each peptide, was found an unidentified amino acid residue; all other amino acid residues were in accord with the known sequence. It is thus concluded that Cys71 of M.HaeIII forms a covalent bond to DNA during catalytic methyl transfer. This finding represents a direct experimental verification for the hypothesis that the conserved Cys residue of DCMtases is the catalytic nucleophile [Wu, J. C., & Santi, D. V. (1987) J. Biol. Chem. 262, 4778-4786].(ABSTRACT TRUNCATED AT 250 WORDS)

Amines↗

PubMed1991

Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-beta receptor system.

We describe the primary structure of rat betaglycan, a polymorphic membrane-anchored proteoglycan with high affinity for transforming growth factor-beta (TGF-beta). As deduced from its cDNA sequence, the 853 amino acid core protein of betaglycan has an extracellular domain with clustered sites for potential attachment of glycosaminoglycan chains. These chains are dispensable for TGF-beta binding to the core protein. The transmembrane region and the short cytoplasmic tail of betaglycan are very similar to these regions in human endoglin, an endothelial cell membrane glycoprotein involved in intercellular recognition. The ectodomain of betaglycan can be released as a soluble proteoglycan; a potential cleavage site near the transmembrane region is identical to the highly regulated cleavage site of the membrane-anchored transforming growth factor-alpha precursor. The unique features of betaglycan suggest important roles in cell interaction with TGF-beta.

Amino Acid Sequence↗

PubMed1991

Yeast RNA polymerase II subunit RPB9 is essential for growth at temperature extremes.

The Saccharomyces cerevisiae RNA polymerase II subunit gene RPB9 was isolated and sequenced. RPB9 is a single copy gene on chromosome VII. The RPB9 sequence predicts a protein of 122 amino acids with a molecular mass of 14,200 Da. The yeast RPB9 subunit is similar in size and sequence to a protein encoded by DNA adjacent to the suppressor of the Hairy Wing gene in Drosophila melanogaster. Deletion of the RPB9 gene produced cells that were heat- and cold-sensitive. The RPB9 subunit, like the previously described RNA polymerase II subunit RPB4, is not essential for synthesis of mRNA, but is required for normal cell growth over a wide temperature range.

Amino Acid Sequence↗

PubMed1991

Identification of self peptides bound to purified HLA-B27.

A pool of endogenous peptides bound to the human class I MHC molecule, HLA-B27, has been isolated. Microsequence analysis of the pool and of 11 HPLC-purified peptides provides information on the binding specificity of the HLA-B27 molecule. The peptides all seem to be nonamers, seven of which match to protein sequences in a database search. These self peptides derive from abundant cytosolic or nuclear proteins, such as histone, ribosomal proteins, and members of the 90K heat-shock protein family.

Amino Acid Sequence↗