Murine MHC class Ib gene,<Emphasis Type="Italic"> H2-M2,</Emphasis> encodes a conserved surface-expressed glycoprotein

We have determined the genomic sequence of H2-M2 in seven haplotypes from nine inbred strains of mice and in five wild-derived haplotypes. Except for the spretus haplotype sp1 with a premature stop codon, we found only limited polymorphism. Four of the five amino acid substitutions in the -helices are at positions that would point out from the antigen-binding groove, indicating that the polymorphism might influence receptor recognition rather than antigen binding. The rat homologue, RT1.M2^lv1, has 89% identity to H2-M2 at the nucleotide level and 91% at the amino acid level, and it also encodes an intact MHC class I glycoprotein. Chimeric proteins with ₁₂ or ₃-transmembrane domains encoded by H2-Q9 were detectable on the surface of transfectants with monoclonal antibodies against Qa2, and the full-length M2 protein, labeled by fusion with green fluorescent protein, was detectable with S19.8 monoclonal antibodies. The H2-M2 protein was thus expressed on the cell surface, even in TAP-deficient RMA-S cells at 37 °C, suggesting that it is TAP-independent. We conclude that H2-M2 is a conserved mouse class Ib gene that is translated to a surface-expressed MHC class I molecule with a function still to be elucidated.The nucleotide sequences reported in this paper have been submitted to GenBank with the accession numbers AY302188–AY302217 for all H2-M2 sequences and AY302218 for RT1.M2, AY326271 for RT1.M2-2, and AY327254 for the RT1.M2 microsatellite marker     

Molecular characterization of major histocompatibility complex class 1 (MHC-I) from squirrel monkeys (<Emphasis Type="Italic">Saimiri sciureus</Emphasis>)

Little is known about the major histocompatibility complex (MHC) class 1 in squirrel monkeys (Saimiri sciureus). We cloned, sequenced and characterized two alleles and the cDNA of the coding region of MHC class 1 in these New World monkeys. Phylogenetic analyses showed that these sequences are related to HLA class 1 genes (HLA-A and HLA-G). The structure and organization of one of the two identified clones was similar to that of a class 1 MHC gene (HLA-A2). All the exon/intron splice acceptor/donor sites are conserved and their locations correspond to the HLA-A2 gene. The sequences of the newly described cDNAs reveal that they code for the characteristic class 1 MHC proteins, with all the features thought necessary for cell surface expression. Typical sequences for the leader peptide, ₁, ₂, ₃, transmembrane and cytoplasmic domains were found.The nucleotide sequence data reported in this paper have been submitted to the GenBank database and have been assigned the accession numbers AJ438576 (Sasc-G*31), AJ438577 (Sasc-G*25), AY282760 (Sasc-G*03), AY282761 (Sasc-G*04) and AY282762 (Sasc-G*05). Sequences were named as recommended by Klein and co-workers (1990)     

Action of a cell wall proteinase from Lactococcus lactis subsp. cremoris SK11 on bovine α s1-casein

Summary The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris SK11 was partially purified and incubated with _s1-casein for various times up to 48 h. Sixteen trifluoroacetic acid-soluble oligopeptide hydrolysis products were identified by determination of the aminp acid sequence. Eleven of these oligopeptides originated from the 78-residue sequence comprising the C-terminal region of _s1-casein and were present among the products after the first 60 min of digestion. Three oligopeptides from the N-terminal region and two others from the central region of the _s1-casein sequence were also present among the early digestion products although in smaller amounts than most of the oligopeptides from the C-terminal region. No cleat consensus sequence of amino acid residues surrounding the cleavage sites could be identified.Offprint requests to: G. G. Pritchard     

Inheritance of seed α-amylase inhibitor in the common bean and genetic relationship to arcelin

The inheritance of seed -amylase inhibitor in the common bean and the genetic relationships among the variants and six arcelin variants in the common bean were investigated by crossing between accessions containing different AI and arcelin variants. All seed proteins in parental, F₁ and F₂ seeds from the crosses were examined by Western-blot analysis. All F₁ seeds gave combined AI banding patterns from parents on the blotting membranes. The segregation of F₂ seeds for AI variants indicated that the polypeptides of AI variants were inherited as single co-dominant units. Moreover, AI and arcelin behaved as a single block in crosses, indicating a close linkage relationship between the genes controlling these proteins.     

Sequence of a cDNA coding for a rat class II Aα chain: Extensive DNA and protein sequence identity to H-2 Aα and HLA-DC1 α chains

The rat major histocompatibility complex (RT1-B region) codes for two sets of class II molecules (la antigens) referred to as A and E. Each class II molecule is composed of two glycoprotein chains called the A and A or E and E . Two cDNA clones encoding rat A chains were identified from cDNA derived from rat spleen mRNA using a combination of mRNA selection and colony hybridization techniques. The complete nucleotide sequence of the cDNA insert of one of these cDNA clones, pRIa.2, was determined. This sequence codes for the carboxy-terminal 129 amino acids of the rat A chain and 293 nucleotides of 3 untranslated sequence. The rat A chain was shown to be highly homologous in terms of both protein and DNA sequence identities to HLA-DC and H-2 A chains. Comparison between the coding regions of the cDNA insert of pRla.2 and the corresponding region of a cDNA insert encoding an HLA-DC1 chain showed sequence identities of 85% and 81% at the protein and DNA levels, respectively. Comparison between pRIa.2 and cDNA encoding an H-2 A chain sequence showed identities of 91% for both protein and DNA. Results are discussed which strongly suggest that the class II A and E primordial genes arose by gene duplication prior to the evolutionary divergence of the mammals.     

Three new rare variants of α1-Antitrypsin

Summary Three new rare genetic variants of the serum protein ₁-antitrypsin (₁-protease inhibitor) have been identified in a Caucasian population. The new alleles in the PI system are PI ^*EFRA, PT^*PCAS, and PI ^*XALB. When compared with the normal type M by isoelectric focusing in polyacrylamide, Efranklin (EFRA) is anodal, and Pcastoria (PCAS) and Xalban (XALB) are cathodal. These variants have been compared with previously described variants by isoelectric focusing and by electrophoresis in agarose and acid starch gels. All three variant alleles appear to be associated with normal amounts of ₁-antitrypsin, assayed both by functional and immunological methods.This work was supported by a grant from the Medical Research Council of Canada     

The Population Genetics of the α-2 Globin Locus of Orangutans (Pongo pygmaeus)

In this study, the molecular population genetics of the orangutans -2 globin (HBA2) gene were investigated in order to test for the action of natural selection. Haplotypes from 28 orangutan chromosomes were collected from a 1.46-kilobase region of the -2 globin locus. While many aspects of the data were consistent with neutrality, the observed heterogeneous distribution of polymorphisms was inconsistent with neutral expectations. Furthermore, a single amino acid variant, found in both the Bornean and the Sumatran orangutan subspecies, was associated with different alternative synonymous variants in each subspecies, suggesting that the allele may have spread separately through the two subspecies after two distinct origination events. This variant is not in Hardy–Weinberg equilibrium (HWE). These observations are consistent with neutral models that incorporate population structure and models that invoke selection. The orangutan Plasmodium parasite is a plausible selective agent that may underlie the variation at -2 globin in orangutans.This article contains electronic supplementary material.[Reviewing Editor: Dr. Deborah Charlesworth]Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers AY372078–AY372110.     

Identification of surface exposed amino acids of isolated and membrane-bound CF1 by protease accessibility

In order to compare surface-exposed amino acids in isolated and membrane-bound CF₁ the technique of limited proteolysis was employed. The cleavage sites of several proteases were identified by sequence analysis of the resulting peptides after isolation by SDS-PAGE. In isolated CF₁ the N-terminal region of the subunit was found to be highy sensitive to proteases; the accessible peptide bonds included E17-G18, R21-E22, E22-V23, and K24-V25. Additional protease-attacked bonds in subunit were S86-S87, xE125-S126. and R127-L128. In the subunit of isolated CF₁ the bonds L14-E15 and V76-A77 were identified as being accessible. All identified protease accessible amino acids are located at the protein surface according to a molecular model of CF₁ computed after the crystal structure of mitochondrial F₁ by S. Engelbrecht (1997). In membrane bound CF₁ the primarily accessible peptide bond of the N-terminal domain of is R21-E22. After this bond is cleaved by trypsin, the K24-V25 becomes accessible to further trypsin attack. Moreover, the peptide bonds R14O-S141 and G16O-R161 are cleaved. According to the Engelbrecht model G16O is almost completely shielded and actually this amino acid was hardly accessible to protease in isolated CF₁. The subunit in general is much more sensitive to proteolysis in membrane-bound than in solubilized CF₁. In the subunit of membrane-bound CF₁ a papain-sensitive bond G102-G103 was identified. The results indicate major structural alterations when CF₁ is extracted from the CF₀CF₁ complex. Thiol modulation, i.e. reduction of the regulatory disulfide bond between C199 and C205 of y subunit, enhances the accesibility of a number of peptide bonds, in particular G160-R161, to proteolytic attack by papain. In contrast, thylakoid membrane energization results in masking of this peptide bond.     

Open reading frame sequencing and structure-based alignment of polypeptides encoded by RT1-Bb, RT1-Ba, RT1-Db, and RT1-Da alleles

MHC class II genes are major genetic components in rats developing autoimmunity. The majority of rat MHC class II sequencing has focused on exon 2, which forms the first external domain. Sequence of the complete open reading frame for rat MHC class II haplotypes and structure-based alignment is lacking. Herein, the complete open reading frame for RT1-B, RT1-B, RT1-D, and RT1-D was sequenced from ten different rat strains, covering eight serological haplotypes, namely a, b, c, d, k, l, n, and u. Each serological haplotype was unique at the nucleotide level of the sequenced RT1-B/D region. Within individual genes, the number of alleles identified was seven, seven, six, and three and the degree of amino-acid polymorphism between allotypes for each gene was 22%, 16%, 19%, and 0.4% for RT1-B, RT1-B, RT1-D, and RT1-D, respectively. The extent and distribution of amino-acid polymorphism was comparable with mouse and human MHC class II. Structure-based alignment identified the 65–66 deletion, the 84a insertion, the 9a insertion, and the 1a–1c insertion in RT1-B previously described for H2-A. Rat allele-specific deletions were found at RT1-B76 and RT1-D90–92. The mature RT1-D polypeptide was one amino acid longer than HLA-DRB1 due to the position of the predicted signal peptide cleavage site. These data are important to a comprehensive understanding of MHC class II structure-function and for mechanistic studies of rat models of autoimmunity.Nucleotide sequence data reported are available in the GenBank database under the accession numbers AY626180–AY626189 for all RT1-Bb sequences, AY626190–AY626199 for all RT1-Ba sequences, AY626200-AY626209 for all RT1-Db sequences and AY626210–AY626219 for all RT1-Da sequences.     

The influence of ethanol on the functional status of GABA(A) receptors

Gamma aminobutyric acid (GABA) is one of the main inhibitory neurotransmitters in the mammalian brain. Its effects are realized via GABA_A, GABA_B, and GABA_C receptors. GABA_A is the most abundant type of GABA receptors. It consists of six classes of subunits, , , , , , and . Acute and chronic exposures to ethanol are accompanied by changes in structure and function of GABA_A receptors. These changes may be a basis for altered behavior seen in alcoholism.     

Heterologous expression of metabotropic glutamate receptor subtype 1 in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The upstream region of the isocitrate lyase gene (UPR-ICL) from the n-alkane-utilizing yeast Candida tropicalis serves as a useful promoter of gene expression in the yeast Saccharomyces cerevisiae. The production of rat metabotropic glutamate receptor 1 (mGluR1), which belongs to the G-protein-coupled receptor (GPCR) family, was tested under the control of UPR-ICL. Expression of mGluR1 was found in recombinant clones and enhanced by replacing the signal sequence of mGluR1 with the corresponding region of the -factor receptor (Ste2), which is a GPCR found in S. cerevisiae. Moreover, the membrane fraction from a recombinant clone associated with Vesl-1S/Homer-1a protein binds the mGluR1 in rat cerebellum. These results suggest that the UPR-ICL-controlled gene expression system is useful for heterologous GPCRs in S. cerevisiae.     

Purification, cloning, and properties of α-galactosidase from Saccharopolyspora erythraea and its use as a reporter system

An -galactosidase from the erythromycin-producing bacterium Saccharopolyspora erythraea was purified to near homogeneity. The enzyme has an apparent molecular mass of 45 kDa as determined by SDS-PAGE. The pH optimum, K_m for p-nitrophenyl--d-glucopyranoside (pNPG), K_m for melibiose and the V_max are similar to those of other studied -galactosidase enzymes. The N-terminal amino-acid sequence of this protein was determined. PCR amplification was used to generate a 640-bp product using oligonucleotide primers based on the N-terminal amino-acid sequence and a downstream region that is conserved in other related -galactosidase enzymes. This fragment was used as a probe to clone the -galactosidase gene, designated melA, from a S. erythraea lambda phage chromosomal library. S. erythraea appears to possess an unique -galactosidase enzyme, encoded by melA, that can utilize galactopyranosides as carbon sources. Furthermore, the ability to use the product of melA as a reporter enzyme in S. erythraea has been demonstrated. The -galactosidase uses the substrates 5-bromo-4-chloro-3-indoyl--d-galactosidase (X--gal) on agar media and pNPG in liquid media.     

The GABAA receptor family in the mammalian brain

The GABA_A-benzodiazepine receptor protein from bovine brain was purified by affinity chromatography and the subunit composition examined by gel electrophoresis in sodium dodecyl sulfate. Protein staining revealed a doublet at 51–53 kDa, a band at 55 kDa, and a broad band at 57–59 kDa. The 51 and 53 kDa bands co-migrated with the ₁ and ₂ gene products identified by Western blotting with subtype-specific antibodies. These two bands were also photoaffinity labeled by [³H]flunitrazepam, as was a breakdown product at 44 kDa. Partial sequencing of proteolytic fragments of these polypeptides yielded sequences found in all clones, and identified the benzodiazepine binding site within residues 8–297 and probably between 106–297 of ₁; the 44 kDa and 31 kDa bands yielded fragments containing ₃ sequence. The native ₃ polypeptide was identified with subtype-specific antibody at 57 kDa overlapping with the two major bands photolabeled with [³H]muscimol at 55 and 58 kDa. Antisera to a -selective peptide recognized four bands at 60, 58, 57 and 55 kDa. Thus, one can identify 6–8 distinct polypeptides with the possibility of another 4–6 in purified GABA_A receptor proteins, depending on brain region, consistent with the family of gene products suggested by molecular cloning.Special issues dedicated to Dr. Eugene Roberts     

Molecular cloning,sequencing and expression of an α2-adrenergic receptor complementary DNA from rat brain

We have isolated a cDNA clone from rat brain using a human platelet ₂-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney ₂-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5-untranslated region. Southern-blot analysis with a human platelet ₂-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain ₂-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A⁺) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the ₂-adrenergic antagonist [³H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the _2A subclass. We conclude that the rat brain cDNA encodes a new ₂-adrenergic receptor subtype that may be brain-specific.Abbreviations G protein guanine nucleotide-binding proteins - cA2-47 ₂-adrenergic receptor cDNA from rat brain - SSC (1X SSC contains 0.15 M NaCl, 15 mM Na₃citrate, pH 7.0)     

Enhanced expression of GTP-binding proteins in differentiated U937 monocytic cells: Possible involvement of tyrosine kinase and protein kinase C

Monocytic U937 cells were differentiated into mature macrophages in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h at 37°C. We investigated the alterations in the expression of GTP-binding proteins that take place during differentiation of these cells. A 40 KDa -subunit of the inhibitory G-protein was identified by specific antibodies to G_i-1/2 and G_i-3 on Western blots and also by ADP-ribosylation catalyzed by pertussis toxin. The expression of the 40 KDa G_i subunit was increased 3.4 fold in differentiated cells. The expression of a 43 kDa G_s subunit identified by Western blotting using specific antibody to G_s and by ADP-ribosylation in the presence of cholera toxin was increased approximately 2 fold in differentiated cells. A faintly recognizable 46 KDa G_s subunit was also increased but to a lesser extent (1.3 fold). Small molecular weight GTP-binding proteins identified by [³⁵S]GTPS binding on nitrocellulose blots were also increased significantly. The PMA-induced expression of G_i-1/2 and G_s subunits was blocked to control level by both genistein and staurosporine, inhibitors of protein tyrosine kinase and protein kinase C, respectively. However, staurosporine was unable to block the PMA-induced expression of G_i-3; this was blocked only by genistein. These data suggest a role for tyrosine kinase and protein kinase C in the expression of G-proteins during differentiation of U937 cells.     

Cloning and nucleotide sequence of the α-galactosidase cDNA from Cyamopsis tetragonoloba (guar)

Polyadenylated mRNA was purified from the aleurone cells of Cyamopsis tetragonoloba (guar) seeds germinated for 18 h and used for the construction of a cDNA library. Clones with the -galactosidase encoding gene were identified using oligo-nucleotide mixed probes based on the NH₂ terminal amino acid sequence and on the sequence of an internal peptide. The nucleotide sequence of the cDNA clone showed that the enzyme is synthesized as a precursor with a 47 amino acid NH₂ terminal extension. This pre-sequence most likely functions to target the protein outside the aleurone cells into the endosperm. Based upon structural features, it is proposed to divide the precursor into a pre-(signal sequence) part and a glycosylated pro-part comparable with those of the yeast mat A/ factor and killer factor. A comparison of the derived amino acid sequence of this -galactosidase from plant origin revealed significant stretches of homology with respect to the amino acid sequences of the enzymes from Saccharomyces cerevisiae and from human origin but only to a minor extent compared with the -galactosidase from Escherichia coli.     

DNA sequence analysis of the rat RT1. B α gene

The major histocompatibility complex (MHC) of the rat (RT1 complex) encodes two sets of class II molecules referred to as RT1.B and RT1.D. The RT1. B gene was isolated for a Sprague-Dawley (RT1^b) rat genomic library using a rat RT1.B chain cDNA as a hybridization probe. The coding and the majority of the intron DNA sequence was determined. The structure of the RT1. B gene is equivalent to that of H-2 and HLA chain genes. Comparison of the nucleotide and predicted amino acid sequences of the RT1.B gene with those of the H-2 and HLA genes revealed a high degree of overall sequence conservation. However, two regions of the first external domain (l), residues 19–23 and 45–78, exhibit marked sequence variation. Two blocks of conserved nucleotide sequence were identified in the 5 promoter region of the RT1. B gene that have been described in all MHC class II genes sequenced to date. These conserved sequences may be involved in the coordinate regulation of expression of class II genes. The cloned RT1.B gene was efficiently transcribed when transfected to mouse L cells.     

Chromosomes in old age: A six year longitudinal study

Summary A new variant of ₁-antitrypsin has been detected with the aid of isoelectric fucosing on polyacrylamide slab gels. In contrast with many other variants this new ₁-antitrypsin allele is found in 10–15% of the phenotypes examined. The electrophoretic properties of the new ₁-antitrypsin variant in isofocusing polyacrylamide gels differ only silightly from the most common ₁-antitrypsin allele M. On the basis of its electroforetic behaviour we propose the term M_N to indicate this new protease inhibitor variant. The isofocusing technique employed, provides an easy to handle, very reproducible method for determining the ₁-antitrypsin phenotype and can be employed for large scale screening.     

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent andcAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase

Ca,phospholipid-dependent (PKC) andcAMP-dependent (PKA) protein kinases phosphorylate the -subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol³²P/mol of -subunit, respectively. PKA (in contrast to PKC) phosphorylates the -subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that³²P is incorporated into the N-terminal 41-kDa fragment of the -subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the -subunit molecule. These findings suggest that PKC phosphorylates the -subunit of the Na,K-ATPase within the region restricted by C₃ and T₁ cleavage sites.     

Functional assessment of genetic variants of α1-Antitrypsin

Summary The specific activity of thirteen genetic variants of the protease inhibitor ₁-antitrypsin (₁-AT) has been determined. Elastase inhibitor activity was assayed using protein substrates (elastin and gelatin) and the synthetic substrate N-tert-butoxycarbonyl-l-alanine p-nitrophenyl ester. The synthetic substrate -N-benzoyl-dl-arginine p-nitroanilide HCl was used to assay trypsin inhibitor activity. The specific activity of ₁-AT was expressed as serum inhibition/immunological concentration of ₁-AT. Sera of PI type FM had reduced specific activity with elastase, but not with trypsin. With the possible exception of MP, no other variants showed significant differences in specific activity when compared with normal PI type M.This research was supported by the Medical Research Council of Canada (No. MA 5426)