Secretion of a soluble class I molecule encoded by the Q10 gene of the C57BL/10 mouse

The DNA sequence of the Q10 genes appears to be highly conserved amongst strains of mice and has only been found to be transcribed in the liver. An examination of the nucleotide sequence of the exon that normally encodes the transmembrane domain of class I molecules suggested that the Q10 gene encodes a secreted protein. We have established this by showing that L cells transformed with an expression vector containing the Q10 gene secrete a class I molecule which was identified with an antiserum raised against a peptide predicted by the Q10 transmembrane exon. Both the L cell-derived Q10 molecule and a class I protein immunoprecipitated from serum with this anti-peptide antiserum have mol. wts. of approximately 38 000; the Q10 molecule secreted by L cells is heterogeneous in mol. wt. This heterogeneity was drastically reduced after endoglycosidase F treatment, suggesting that Q10 molecules secreted into the serum by the liver may be glycosylated differently from those secreted by L cells. Endoglycosidase F treatment of both the L cell and serum forms of the soluble molecule yielded two products with mol. wts. of approximately 32 000 and 35 000; this is consistent with the observation that the predicted Q10 protein sequence has two potential glycosylation sites. In contrast to previous published results, the Q10 molecule reacted with rabbit anti-H-2 antisera which is consistent with its greater than 80% homology to the classical transplantation antigens.     

Sequence of the Clostridium thermocellum mannanase gene man26B and characterization of the translated product

The man26B gene of Clostridium thermocellum strain F1 was found in pKS305, which had been selected as a recombinant plasmid conferring endoglucanase activity on Escherichia coli. The open reading frame of man26B consists of 1,773 nucleotides encoding a protein of 591 amino acids with a predicted molecular weight of 67,047. Man26B is a modular enzyme composed of an N-terminal signal peptide and three domains in the following order: a mannan-binding domain, a family 26 mannanase domain, and a dockerin domain responsible for cellulosome assembly. We found that this gene was a homologue of the man26A gene of C. thermocellum strain YS but that there were insertion or deletion mutations that caused a frame-shift mutation affecting a stretch of 26 amino acids in the catalytic domain. Man26B devoid of the dockerin domain was constructed and purified from a recombinant E. coli, and its enzyme properties were examined. Immunological analysis indicated that Man26B was a catalytic component of the C. thermocellum F1 cellulosome.     

Preimplantation mouse embryos and liver express the same type I keratin gene product

The cytoskeletal B protein isolated from extraembryonic endodermal cells (Endo B) is a 50-kDa subunit of intermediate filaments that is expressed in trophoblast and extraembryonic endoderm of early mouse embryos. Endo B was compared to cytokeratin D of adult mouse liver by immunoprecipitation, two-dimensional gel electrophoresis, and peptide mapping. The two proteins were indistinguishable. A cDNA probe for Endo B mRNA identified mRNA species of similar size in liver and endoderm, and primer extension analysis indicates that the Endo B mRNAs from the two cell types have similar 5' ends. An internal fragment of the Endo B cDNA was found to cross-hybridize with a conservative domain of a human type I keratin cDNA under low stringency conditions, demonstrating that Endo B is related to type I keratins. However, under stringent conditions necessary for genomic Southern analysis, mouse and human genomic fragments homologous to the Endo B cDNA were distinct from those defined by hybridization with the type I keratin cDNA. These results indicate that Endo B is related to the type I keratin family and expands the number of type I keratin genes identified in both the mouse and human genomes. It is likely that extraembryonic endoderm, one of the first differentiated cell types of the mammalian embryo, and adult liver express the same Endo B gene.     

Cloning of the Bacillus firmus OF4 cls gene and characterization of its gene product

The gene that codes for cardiolipin (CL) synthase and an adjacent gene that codes for a MecA homolog in the alkaliphilic bacteria Bacillus firmus OF4 have been cloned and sequenced (GenBank accession number U88888). The cls gene contains 1509 nucleotides, corresponding to a polypeptide of 57.9 kDa. The predicted amino acid sequence has 129 identities and 100 similarities with the Escherichia coli CL synthase. Homologies were also noted with polypeptide sequences from putative cls genes from Bacillus subtilis and Psuedomonas putida. Conserved histidine, tyrosine, and serine residues may be part of the active site and participate in phosphatidyl group transfer. The B. firmus OF4 cls gene product was inserted into plasmid pET3 to form a recombinant plasmid pDG2, which overproduces CL synthase in E. coli. A membrane fraction containing the overproduced enzyme converts phosphatidylglycerol to CL and glycerol. The B. firmus enzyme is stimulated by potassium phosphate, inhibited by CL and phosphatidate, and has a slightly higher pH optimum than the E. coli enzyme.     

A soluble class I molecule analogous to mouse Q10 in the horse and related species

Horse serum is shown to contain a soluble class I molecule analogous to the secreted Q10 molecule in the mouse. This molecule has several similarities to the recently described mouse Q10 molecule: (1) it is smaller than membrane-bound equine class I molecules; (2) it occurs in a high molecular mass complex of 200–300 kd in serum; and (3) the serum levels of the equine molecule are similar to that of the Q 10 molecule (about 30 g/ml). A soluble molecule is also detected in the sera of species related to the horse; it has in fact been found in all the wild members of the order Perissodactyla so far tested. However, it was not detected in the serum of members of the orders Carnivora, Sirenia, Proboscidea, Artiodactyla, and Primates that were tested, nor in the serum of members of the order Rodentia other than in that of the genus Mus. Abbreviations used in this paper ₂m beta-2 microglobulin - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Identification of two major histocompatibility complex class Ib genes, Q7 and Q9, as the Ped gene in the mouse

The Ped (preimplantation embryonic development) gene influences the rate of preimplantation embryonic development and subsequent embryonic survival. The protein product of the Ped gene, the Qa-2 protein, is a major histocompatibility complex (MHC) class Ib protein. There are two alleles of the Ped gene, fast (Qa-2 [+]) and slow (Qa-2 [-]). Qa-2 is encoded by four very similar MHC class Ib genes: Q6, Q7, Q8, and Q9. Recent research in our laboratory has shown that the Ped phenotype is potentially encoded by the Q7 and/or Q9 gene because the Q7 and Q9 genes, but not the Q6 or Q8 gene, are expressed during preimplantation mouse embryonic development. In this study we utilized microinjection of transgenes to assess the functional roles of both the Q7 and Q9 genes in control of the rate of preimplantation development. The Q7 gene, the Q9 gene, and a combination of the Q7 and Q9 genes were microinjected into Ped slow zygotes, and the Ped phenotype and cell surface expression of Qa-2 protein were assayed after a 72-h or 96-h incubation period. We found that the microinjected individual Q7 and Q9 genes increased the rate of preimplantation development. Simultaneous injection of the Q7 and Q9 genes did not have a synergistic effect on the Ped phenotype. Microinjection of the Q7 and/or Q9 genes resulted in protein expression in 10-25% of the microinjected embryos. These results show that both the Q7 and Q9 genes encode the mouse Ped phenotype.     

CML characterization of a product of a second class I locus in the rat MHC

In the rat, genes that control the expression of target antigens detected by cell-mediated lympholysis (CML) are present in the major histocompatibility complex (MHC). The relationship of these loci, CT and Ag-L, to each other and to other loci within the MHC is unknown. In this report, we demonstrate the existence of a CML target antigen in the (DA × BN)F₁ anti-DA.11(BI) strain combination. The gene coding for this antigen is linked to the RT1 complex as indicated by the CML reactivity of targets from backcross and congenic animals. Inhibition studies demonstrated that this antigen has the widespread tissue distribution characteristic of class I antigens, and the gene coding for this CML antigen maps coincident with the RT1.E class I locus as indicated by the lysis of targets from the recombinant strains r10 and r11. The CML can be blocked by antisera directed against a product of the RT1.E locus. The locus controlling this CML reactivity, like CT and Ag-L, has been separated from RT1.A by recombination; unlike CT and Ag-L, the product of this CML locus appears to be identical with an RT1.E allelic product that has been serologically identified and biochemically characterized.Abbreviations used in this paper MHC major histocompatibility complex - CML cell-mediated lympholysis - Con A concanavalin A - SD standard deviation - HEPES N-2-hydroxy-piperazine-N-2-ethanesulfonic acid - CPM counts per minute - grc growth and reproduction complex     

Sequence,expression, and mapping of a rat Mhc class I b gene

The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence databased and have been assigned the accession number X70066.     

cDNA encoding the chicken ortholog of the mouse dilute gene product. Sequence comparison reveals a myosin I subfamily with conserved C-terminal domains.

We report the cDNA-deduced primary structure of the chicken counterpart of the murine dilute gene product, a member of the myosin I family. Comparison of the chicken and mouse sequences reveals a distinct pattern of domains of high and low sequence conservation. An internal deletion of 25 amino acids probably reflects differential mRNA processing. Compared with other myosin heavy chain molecules, sequence similarity is highest with the MYO2 gene product of Saccharomyces cerevisiae. The MYO2 protein, implicated in vectorial vesicle transport, is homologous to the dilute protein over practically its entire length. In addition, the C-terminal domain of the dilute protein is highly similar to a putative glutamic acid decarboxylase sequence cloned from mouse brain. Alternatively, this closely related clone might represent an isoform of the dilute protein derived from a second gene, potentially involved in genetic conditions related to dilute.     

Mhc class I and non-class I gene organization in the proximal H2-M region of the mouse

 A bacterial artificial chromosome (BAC) contig was constructed across the proximal part of the H2-M region from the major histocompatibility complex (Mhc) of mouse strain 129 (H2 ^bc ). The contig is composed of 28 clones that span approximately 1 megabasepair (Mb), from H2-T1 to Mog, and contains three H2-T genes and 18 H2-M genes. We report the fine mapping of the H2-M class I gene cluster, which includes the previously reported M4-M6, the M1 family, the M10 family, and four additional class I genes. All but two of the H2-M class I genes are conserved among haplotypes H2 ^k , H2 ^b , and H2 ^bc , and only two genes are found in polymorphic HindIII fragments. Six evolutionarily conserved non-class I genes were mapped to a 180 kilobase interval in the distal part of the class I region in mouse, and their order Znf173-Rfb30-Tctex5-Tctex6-Tctex4-Mog was found conserved between human and mouse. In this Znf173-Mog interval, three mouse class I genes, M6, M4, and M5, which are conserved among haplotypes, occupy the same map position as the human HLA-A class I cluster, which varies among haplotypes and is diverged in sequence from the mouse genes. These results further support the view that class I gene diverge and evolve independently between species. Received: 27 April 1998 / Revised: 4 June 1998     

Sequence and structure of the mouse connexin45 gene

The connexin45 (Cx45) gene was cloned from a mouse genomic Bacterial Artificial Chromosome library. Approximately 8.4 kb of the genomic DNA was sequenced, and the structure of the Cx45 gene was determined. The mouse Cx45 gene is composed of 3 exons, with the entire coding sequence contained within exon III (EMBL Accession Number AJ300716). This structure is unique for the Cx45 gene, since all other members of the connexin family have only two exons. In addition, computer analysis reveals a potential TATA box and two putative AP-1 binding sites in the 5 region of the gene. Sequence alignment with connexin43 indicates substantial homology in the intronic sequences upstream of the 3 exons of the two genes, suggesting that the Cx45 gene is inherently similar to the rest of the connexin family, and that it probably evolved from an ancestor common to the other connexins.     

Different regulation of class I gene expression in the adult mouse and during development.

The expression of the class I genes encoding for histocompatibility Ag is complex both in adult and during development. Although ubiquitously expressed in the adult, the mRNA level of class I genes is variable from one organ to another. During development, H-2K mRNA expression has two phases: the first from blastocyst to day 11, where H-2K mRNA level is extremely low, and the second, beginning after day 11, when H-2K mRNA expression increases first dramatically (10x) and then progressively to birth. To localize the sequences responsible for the regulation of H-2K gene expression in the adult and during development, we have constructed a series of transgenic strains carrying 1) a 9-kb native H-2K gene, H-2K LF, corresponding to the entire H-2Kb gene with 2 kb of upstream sequences and 3 kb of downstream sequences, and 2) two hybrid constructs linking the same 5'-flanking region of H-2Kb gene to two reporter genes, the human growth hormone and the human c-myc proto-oncogene. Expression of the transgenes was compared with that of the endogeneous H-2K gene in adult organs and during development of the different transgenic strains. In the adult, the three constructs behave almost like the endogeneous H-2K gene, but the H-2K LF construct is the only one whose expression is independent of the integration site and related to the copy number. During development, both fusion genes are barely expressed in the embryo as well as in the extra-embryonic tissues, whereas the H-2K LF transgene expression parallels that of the endogeneous class I gene. Therefore, our results show that H-2K developmental regulatory sequences are not included in the region that controls H-2K mRNA expression in the adult, indicating that H-2K class I gene expression in adult organs and in development is regulated by different mechanisms.