Halobacterial flagellins are encoded by a multigene family. Characterization of five flagellin genes

Purified flagellar filaments of Halobacterium halobium contain three different protein species based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These proteins were designated as flagellins Fla I, Fla II, and Fla III and were characterized as sulfated glycoproteins with N-glycosidically linked oligosaccharides of the type GlcA-(1----4)-GlcA-(1----4)-GlcA-(1----4)-Glc. All halobacterial flagellin polypeptides are immunologically cross-reactive. A gene fragment of one flagellin was isolated in an expression vector using antibody probes. Using this gene fragment as probe, we identified, subcloned, and determined the nucleotide sequences of five different but highly homologous flagellin genes. Two flagellin (flg) genes are arranged tandemly at one locus (flg A1 and -2), and the other three in a tandem arrangement at a different locus (flg B1, -2, and -3), Two flg mRNAs were detected, one from the A genes and the other from the B genes. Based on immunological analysis, the products of the flg A1 and A2 are Fla II and Fla I, respectively.     

Homology maps of the Drosophila alpha-tubulin gene family: one of the four genes is different.

We describe the intron-exon structure of and the homology among the four alpha-tubulin genes of Drosophila melanogaster. Three of the genes share a highly conserved 1.3 kb sequence which corresponds to most of the RNA complementary portion of the genes. The fourth gene is different. Its 5' half has weak homology and its 3' half has moderate homology to the other three genes. The homology maps were first determined by electron microscopy of heteroduplexes between pairs of genes. Higher resolution maps were then obtained by gel analysis of heteroduplexes that had been digested with S1 nuclease.     

Halobacterial flagellins are encoded by a multigene family. Identification of all five gene products

Flagellins of Halobacterium halobium are encoded in five different but homologous genes. Flagellins isolated from purified flagella were digested and the resulting peptides sequenced. The amino acid sequence data obtained prove that all five gene products are expressed and integrated into the flagellar bundle.     

Biochemical characterization of polypeptides encoded by mutated human Ha-ras1 genes.

We expressed six forms of p21-ras polypeptides in Escherichia coli with differing transformation potentials resulting from amino acid substitutions at position 12. The ability of the encoded p21's to autophosphorylate, bind guanine nucleotides, and hydrolyze GTP was assessed. All versions of p21 bound GTP equivalently; the kinase activity, while dependent upon residue 12, did not correlate with the transforming potential of the polypeptide. All transforming versions exhibited an impaired GTPase activity, while a novel nontransforming derivative [p21(pro-12)] possessed an enhanced GTPase activity. These results provide strong support for the proposal that an impairment of the cellular p21 GTPase activity can unmask its transforming potential.     

Conserved sequence motifs upstream from the co-ordinately expressed vitellogenin and apoVLDLII genes of chicken.

The vitellogenin and apoVLDLII yolk protein genes of chicken are transcribed in the liver upon estrogenization. To get information on putative regulatory elements, we compared more than 2 kb of their 5' flanking DNA sequences. Common sequence motifs were found in regions exhibiting estrogen-induced changes in chromatin structure. Stretches of alternating pyrimidines and purines of about 30-nucleotides long are present at roughly similar positions. A distinct box of sequence homology in the chicken genes also appears to be present at a similar position in front of the vitellogenin genes of Xenopus laevis, but is absent from the estrogen-responsive egg-white protein genes expressed in the oviduct. In front of the vitellogenin (position -595) and the VLDLII gene (position -548), a DNA element of about 300 base-pairs was found, which possesses structural characteristics of a mobile genetic element and bears homology to the transposon-like Vi element of Xenopus laevis.     

Rabbit variable kappa light chain regions: subgroups contain polypeptides encoded by multiple genes.

Two identical light chain variable regions were identified in anti-streptococcal Group A-variant antibodies elicited in litter-mate rabbits by hyperimmunization with vaccine. In addition, one rabbit produced two additional clonally restricted antibodies to this polysaccharide antigen. The partial amino acid sequence of the light chain of one of these antibodies was identical with the dominant antibody light chain sequence, while the light chain of the other antibody, also partially established, showed significant variations in the framework-associated regions with identical CDRI and II. Since all of these light chains were from a small subset of rabbit kappa light chain pools (b4 allotype) the data suggest, together with other light chains reported in the literature, that more than one copy of variable region genes are present in the germ-line per subgroup. Furthermore, framework associated amino acid substitutions are not random; this suggests the existence of some "ordered" mechanism for linked amino acid substitutions (presumably recombination). Furthermore one light chain can pair with more than one heavy chain to yield functional antibodies.     

A family of related proteins is encoded by the major Drosophila heat shock gene family.

At least four proteins of 70,000 to 75,000 molecular weight (70-75K) were synthesized from mRNA which hybridized with a cloned heat shock gene previously shown to be localized to the 87A and 87C heat shock puff sites. These in vitro-synthesized proteins were indistinguishable from in vivo-synthesized heat shock-induced proteins when analyzed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of the pattern of this group of proteins synthesized in vivo during a 5-min pulse or during continuous labeling indicates that the 72-75K proteins are probably not kinetic precursors to the major 70K heat shock protein. Partial digestion products generated with V8 protease indicated that the 70-75K heat shock proteins are closely related, but that there are clear differences between them. The partial digestion patterns obtained from heat shock proteins from the Kc cell line and from the Oregon R strain of Drosophila melanogaster are very similar. Genetic analysis of the patterns of 70-75K heat shock protein synthesis indicated that the genes encoding at least two of the three 72-75K heat shock proteins are located outside of the major 87A and 87C puff sites.     

A survey of genes in the Atlantic salmon (Salmo salar) as identified by expressed sequence tags

We describe the construction and quality analysis of six cDNA libraries from the liver, ovary, testis, brain, spleen and muscle tissues of adult Atlantic salmon. The cDNA libraries were then screened with total cDNA probes to catalogue clones representing the abundant and rare mRNA populations in each tissue. Subsequently, the 5'-terminal DNA sequences of 1152 cDNA clones, composed of 96 clones from each of the abundant and rare mRNA populations in the six tissues, were determined. Bioinformatic analysis revealed that 510 (50%) of the salmon expressed sequence tags (ESTs) of sufficient length showed significant homology to previously identified genes from salmonid and other species, while 517 (50%) of salmon ESTs were unidentified or novel. After accounting for multi-EST redundancy, the 510 identified ESTs provided DNA sequence markers for 178 salmon genes which are listed in terms of tissue of origin and mRNA abundance class.     

Deduced sequence of a malate synthase polypeptide encoded by a subclass of the gene family

We analyzed five malate synthase cDNA clones from the higher plant Brassica napus L. We determined the complete mRNA sequence and showed that the longest cDNA clone, pMS1, contains the entire protein coding region. The deduced polypeptide consists of 561 amino acids with a molecular mass of 63,700 daltons. To discern whether the cloned mRNAs represent distinct malate synthase polypeptides, we compared restriction maps and partial nucleotide sequence of the cDNA clones as well as their pattern of hybridization with restriction fragments in nuclear DNA. The results suggest that the five cloned mRNAs are encoded by either a single gene or by highly conserved members of the gene family.     

Relationship between proteins encoded by three human gamma-crystallin genes and distinct polypeptides in the eye lens.

Although individual gamma-crystallins from the human eye lens have not been successfully purified and sequenced, most of the genes coding for these lens-specific structural proteins have been cloned and characterized. To investigate the relationship between these genes and the gamma-crystallins of the human lens, we made use of mouse cell lines which contain stably integrated copies of the coding sequences for three of the human gamma-crystallin genes coupled to the human metallothionein IIA promoter. The proteins produced by these hybrid genes in cell culture were detected immunologically and compared by physical characteristics with the gamma-crystallins from the human lens. The protein encoded by the G3 gene showed properties identical to those of the 21,000-molecular-weight gamma-crystallin from 11-month-old lens. The protein isolated from the cells expressing the G4 gene was similar to a 19,000-molecular-weight lens gamma-crystallin, while gene G5 encodes a highly basic gamma-crystallin which may be synthesized in only limited amounts in the human lens. These correlations provide a basis for future investigations on the relationship between putative mutations in human gamma-crystallin genes and altered proteins in hereditary lens cataracts.     

Vertebrate histone genes: nucleotide sequence of a chicken H2A gene and regulatory flanking sequences.

The DNA sequence of a chicken genomal fragment containing a histone H2A gene has been determined. It contains extensive 5' and 3' flanking regions and encodes a protein identical in sequence to the histone H2A protein isolated from chicken erythrocytes. In the 5' flanking region, a possible "TATA box" and three possible "cap sites" can be recognised upstream from the initiation codon. To the 5' side of the "TATA box" is found an unusual sequence of 21 A's interrupted by a central G residue. It occupies the same relative position as the P. miliaris H2A gene-specific 5' dyad symmetry sequence and the "CCAAT box" seen in other eukaryotic polymerase II genes but is clearly different from both. A significant feature of the 3' non-coding region is the presence of a 23 base-pair sequence that is nearly identical to a conserved region found in sea urchin histone genes. The coding region is extremely GC rich, with strong selection for these bases in the third position of codons. Not a single coding triplet ends in U. No intervening sequences were found in this gene.     

Nucleotide sequence of a chicken vitellogenin gene and derived amino acid sequence of the encoded yolk precursor protein

The gene encoding the major vitellogenin from chicken has been completely sequenced and its exon-intron organization has been established. The gene is 20,342 base-pairs long and contains 35 exons with a combined length of 5787 base-pairs. They encode the 1850-amino acid pre-peptide of vitellogenin, which is the precursor of the mature yolk proteins, the serine-rich and heavily phosphorylated phosvitin and the lipovitellin. The 217-amino acid phosvitin polypeptide occupies an internal position (residue 1112 through 1328) within the vitellogenin molecule. The 125,000 and 30,000 Mr lipovitellin polypeptides are encoded by the sequences at the N-terminal and the C-terminal sides of the phosvitin section, respectively. The main features of the gene and protein sequences, and the evolutionary implications, are discussed.     

Nucleotide sequence comparison of five human pepsinogen A (PGA) genes: evolution of the PGA multigene family

To unravel the genetic basis for the pepsinogen A (PGA) protein polymorphism, we have isolated and characterized a number of PGA genes, distinguishable by polymorphic EcoRI fragments of 12.0, 15.0, and 16.6 kb. Using a HindIII or AvaII polymorphism, we can discriminate between different 15.0 (15.0 and 15.0*) and 12.0 (12.0s and 12.0l) genes, respectively. The coding sequences of a 15.0 and a 16.6 gene were determined, together with considerable stretches of the 5'- and 3'-flanking regions and introns. The genes were demonstrated to encode Pg5 and Pg4, respectively. Because substitutions in codons 43 and 207 appeared to be critical in the determination of the encoded proteins, we sequenced only these regions in the two 12.0 genes and the 15.0* gene. On the basis of these partial sequences, we assume that these genes encode Pg3. In the evolutionary model of the PGA gene cluster presented here, the 12.0 genes arose by an unequal, but homologous crossover. The results of sequence analysis of the second intron of the 12.0s, 12.0l, 15.0, and 16.6 genes suggest that the two 12.0 genes have arisen from two different crossover events.     

Characterization of the major immediate-early polypeptides encoded by murine cytomegalovirus.

The immediate-early (IE) infected cell proteins induced by the murine cytomegalovirus (Smith strain) were studied. These polypeptides were identified as IE proteins by their synthesis in the presence of actinomycin D after removal from a protein synthesis block mediated by cycloheximide. By using a murine antiserum against murine cytomegalovirus, three abundant polypeptides of 89, 84, and 76 kilodaltons (kd) were immunoprecipitated. The three major proteins are phosphorylated but not glycosylated and share antigenic determinants recognized by monoclonal antibodies. The 84 and 76-kd polypeptides represent post-translational modification products of the 89-kd protein. Accordingly, in vitro translation of IE infected cell RNA revealed only the 89-kd polypeptide. The viral origin of the RNA species directing the synthesis of the major 89-kd IE polypeptide was verified by hybrid selection of IE RNA with DNA fragments representing the region from 0.769 to 0.815 map units of the murine cytomegalovirus genome. IE polypeptides were found to be located in the nuclei and the cytoplasm of infected cells. Studies on the kinetics of IE polypeptide synthesis revealed negative regulatory effects on IE gene expression correlated with the synthesis of early proteins.     

The chicken delta-crystallin gene family. Two genes of similar structure in close chromosomal approximation

delta-Crystallin is a major protein product of the differentiated chicken lens. We have isolated two, non-allelic delta-crystallin genes using a recombinant bacteriophage/chicken genomic DNA library. There appear to be only these two delta-crystallin genes in the haploid chicken genome. Southern hybridization and R-loop analyses indicate that the two genes are oriented on the chromosome with similar 5'-3' polarity. delta 1, arbitrarily designated as the directionally 5' of the two genes, is 6.7 kilobases in length, while delta 2 is 9.2 kilobases. The two delta-crystallin genes are about 4.2 kilobases apart. Structurally, both genes are arranged in a similar and characteristic pattern of 17 exons/16 introns, as judged by electron microscopy. The delta-crystallin gene locus represents a simple model for the study of structural co-evolution and/or functional co-expression of two related genes within a developmentally modulated region of the genome.