Characterization of two novel γ-gliadin genes encoded by K genome of Crithopsis delileana and evolution analysis with those from Triticeae

By acid polyacrylamide gel electrophoresis (A-PAGE) analysis, it was indicated that the electrophoresis mobility of gliadins from Crithopsis delileana (Schult) Roshev (2n=2x=14, KK) had obvious difference with those from common wheat in α, γ and ω region. Using homologous primers, two γ-gliadin genes (gli-Kr1 and gli-Kr2) were isolated from C. delileana, which had been deposited in the GenBank under accession numbers EU283818 and EU283821, respectively. Two γ -gliadin genes of C. delileana had the similar primary structures to the corresponding gene sequences from other wheat related species. The differences were mainly resulted from substitutions, insertions and deletions involving single amino acid residues or motifs of γ-gliadins. The repetitive domains of gli-Kr1 and gli-Kr2 from C. delileana are shorter than most of other sequences. By the alignment of γ-gliadin genes from A, B, D, A^m, A^u, S, S^l, S^sh, S^s and S^b genomes of Triticum and Aegilops, R genome of Secale (γ-secalin), Ee genome of Lophopyrum and K genome of Crithopsis in Triticeae, phylogenetic analysis indicated that two γ-gliadin genes of C. delileana could be clustered together with a γ-gliadin genefrom Ssh genome of Aegilops by an interior paralleled branch. It was the first time that the γ-gliadin genes encoded by K genome of C. delileana were characterized. These could offer precious information for better understanding the qualities associated with gliadins, the response in coeliac disease and studying the evolutionary relationship of gliadins in Triticeae.     

Nucleotide sequences of switch regions of immunoglobulin C epsilon and C gamma genes and their comparison

Immunoglobulin class switch involves a unique recombination event that takes place at the switch (S) region which is located 5' to each constant region (C) gene of the heavy (H) chain. For example, differentiation of the B lymphocyte from a mu-chain producer to an epsilon-chain producer is mediated by the switch recombination between the S mu and S epsilon regions. In order to elucidate the molecular mechanism for the switch recombination, we have determined nucleotide sequences surrounding the class switch recombination sites of the C epsilon and C gamma 3 genes and those in the 5' flanking regions of the C gamma 2a and C delta genes. The results indicate that the 5' flanking regions of all the CH genes except for the C delta gene contain the S regions which comprise tandem repetition of short unit sequences in agreement with the previous analyses of the S gamma 1, S gamma 2b, S mu, and S alpha regions. Comparison of the nucleotide sequences of all the S regions revealed that length as well as nucleotide sequences of the S regions vary among different classes of the CH gene, but they share short common sequences, (G)AGCT and TGGG(G). The nucleotide sequence of the S mu region is homologous to those of the other S regions in the decreasing order of the S epsilon, S alpha, S gamma 3, and (S gamma 1, S gamma 2b, s gamma 2a) regions. We have compared the nucleotide sequences immediately adjacent to the recombination sites of seven rearranged genes and have always fund tetranucleotides TGAG and/or TGGG, except for one case. Such tetranucleotides may constitute a part of the recognition sequence of a putative recombinase. These results provide further support for our previous proposal that the switch recombination may be facilitated by short common sequences dispersed in all the S regions.     

Strict co-linearity of genetic and protein folding domains in an intragenically duplicated rat lens γ-crystallin gene

Two complementary DNA clones pRLγ-2 and pRLγ-3 of different rat lens γ-crystallin messenger RNAs have been used to identify γ-crystallin gene sequences in rat genomic DNA. Subsequently, the DNA present in the 18,000 to 20,000 bases region of the EcoRI digest, giving rise to a strong doublet hybridization signal, was cloned in λ phage Charon-4A. One of the clones, λRCHγ-3, carrying an insert of 17,500 bases has been characterized in detail. From analysis at the restriction enzyme level with 5′-, “middle” and 3′-specific subprobes of pRLγ-3 it could be deduced that λRCHγ-3 contains only one γ-crystallin gene. The coding sequences of this gene are interrupted by intronic DNA. The primary structure of this gene and its flanking regions have been established by sequencing the relevant regions of a subclone of λRCHγ-3, designated pRCHγ-3.1. The sequence data show that the γ-crystallin gene extends over 2700 bases of rat genomic DNA. The gene is split by two introns, one of 87 base-pairs after the third translation codon and a large one of 1880 base-pairs after codon 84. The mosaic structure of the gene is strictly co-linear with the structure of the γ-crystallin polypeptide in that the large intron is positioned in a region which specifies the so-called “connecting peptide” and which links the two highly symmetrical and homologous protein domains. Although expected from the cDNA and protein sequence no introns were observed between the coding regions in the DNA specifying the two homologous folding motifs present in each protein domain. The relevance of this phenomenon in terms of the evolution of the mature γ-crystallin gene is discussed.     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.     

Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis.

The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S₁ nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.     

Production of the human immunoglobulin γ1 chain constant region polypeptides in Escherichia coli

We have determined the nucleotide sequence of the constant region exons of the rearranged human immunoglobulin γ₁ chain gene cloned from a human plasma cell leukemia line, ARH-77. The amino acid sequence deduced from the nucleotide sequence revealed that the allotype of the ARH-77 γ₁ chain was Glm (−1, −2, 3).Recombinant plasmids were then constructed in order to express the human γ₁ chain constant region genes (the Fc region gene and the C_H2-C_H3 domains gene) in Escherichia coli. The human γ₁ chain constant region genes without introns were derived from the genomic gene using synthetic DNA fragments. E. coli carrying each expression plasmid produced antigenically active constant region polypeptides as soluble proteins. In the case of the E. coli-derived Fc region polypeptides, they were generated as monomeric forms in the cytoplasm.     

Identification and characterization of TCRγ and TCRδ chains in channel catfish,Ictalurus punctatus

Channel catfish, Ictalurus punctatus, T cell receptors (TCR) γ and δ were identified by mining of expressed sequence tag databases, and full-length sequences were obtained by 5′-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), diversity (D), joining (J), and constant (C) regions. Three TCRγ V families, seven TCRγ J sequences, and three TCRγ C sequences were identified from sequencing of cDNA. Primer walking on bacterial artificial chromosomes (BACs) confirmed that the TRG locus contained seven TRGJ segments and indicated that the locus consists of (Vγ3-Jγ6-Cγ2)–(Vγ1_n-Jγ7-Cγ3)–(Vγ2-Jγ5-Jγ4-Jγ3-Jγ2-Jγ1-Cγ1). In comparison for TCRδ, two V families, four TCRδ D sequences, one TCRδ J sequence, and one TCRδ C sequence were identified by cDNA sequencing. Importantly, the finding that some catfish TCRδ cDNAs contain TCR Vα-D-Jδ rearrangements and some TCRα cDNAs contain Vδ-Jα rearrangements strongly implies that the catfish TRA and TRD loci are linked. Finally, primer walking on BACs and Southern blotting suggest that catfish have four TRDD gene segments and a single TRDJ and TRDC gene. As in most vertebrates, all three reading frames of each of the catfish TRDD segments can be used in functional rearrangements, and more than one TRDD segment can be used in a single rearrangement. As expected, catfish TCRδ CDR3 regions are longer and more diverse than TCRγ CDR3 regions, and as a group they utilize more nucleotide additions and contain more nucleotide deletions than catfish TCRγ rearrangements.     

Regions of the terminal repetitions of the herpes simplex virus type 1 genome. Relationship to immunoglobulin switch-like DNA sequences

Several recombinant clones isolated from a mouse genomic library were previously shown to hybridize with a SmaI fragment located in the terminal repetition of the S component of herpes simplex virus DNA. We report here the nucleotide sequence of the related regions in two mouse clones, TGL19 and TGL35, as well as that of the SmaI fragment of HSV-1. The mouse DNA clones have a core of repetitive sequences 80% homologous to a tandem repeat (reiteration II) in the viral fragment. The regions of homology are in turn related to immunoglobulin class-switch sequences, due mostly to the presence of the pentamer TGGG(G), involved in class-switch recombination. These results suggest that the HSV genome has recombination sequences identical to those of the host cell and provide a possible explanation for the high frequency of recombination events observed in this region of the viral genome.     

Sequence and expression of the discoidin I gene family in Dictyostelium discoideum

We have isolated recombinant phage and plasmids containing the four developmentally regulated discoidin I genes of Dictyostelium discoideum. Two of the genes are linked within 0.5 × 10³ bases with the same polarity. S¹ nuclease mapping shows that at least three members of the gene family are expressed and that the 5′ ends of the mRNAs start at equivalent sites. The genes have homologous 5′ untranslated regions and extremely divergent 3′ untranslated regions. In addition, some of the genes are flanked by homologous repeat sequences. The genes encode three different isoelectric forms of the protein. Examination of nucleotide sequences in the protein coding region shows that most nucleotide changes in the 5′ half of the gene result in amino acid substitutions while most base substitutions in the 3′ half are neutral.     

Molecular characterization of intact, but cryptic, flagellin genes in the genus Shigella

Flagellin genes (fliC) were detected in two species of the genus Shigella. The fliC_SF gene cloned from Shigella flexneri produced normal-type flagella in an Escherichia coli δ fliC strain while the fliC_SS genes from two Shigella sonnei strains produced curly-type flagella and their expression is repressible by Salmonella FljA repressor. The fliC_SF gene (1650bp) shared high similarity with the E. coli fliC_E gene not only in the 5′ and 3′ constant sequences but also in the upstream and downstream sequences. The fliC_S genes (1572 bp) shared high similarity with the Salmonella typhimurium fliCs gene in the operator and 3’constant sequences and also shared high similarity with the fliC_E gene in the downstream sequence, suggesting that the fliC gene has undergone horizontal transfer and recombination. Differences In nucleotide sequences of the central variable regions among the four fliC genes, including fliC_E and fliC_S, suggest that they started differentiation in each lineage approximately 80 million years ago. Loss of motility in Shigella seems to be evolutionarily a recent event.     

Vaporization of Inorganic Mercury by Cell-free Extracts of Drug Resistant Escherichia coli

The cDNAs encoding venom phospholipase A₂ (PLA₂) inhibitors (PLIs), named Protobothrops elegans (Pe)γPLI-A, PeγPLI-B, PeαPLI-A, and PeαPLI-B, were cloned from the P. elegans liver cDNA library. They were further divided into several constituents due to nucleotide substitutions in their open reading frames. For PeαPLI-A, two constituents, PeαPLI-A_a and PeαPLI-A_b, were identified due to three nonsynonymous substitutions in exon 3. Far-western blot and mass-spectrometry analysis of the P. elegans serum proteins showed the presence of γPLIs, and αPLIs, which can bind venom PLA₂s. In αPLIs from Protobothrops sera, A or B subtype-specific amino acid substitutions are concentrated only in exon 3. A comparison of γPLIs showed that γPLI-As are conserved and γPLI-Bs diversified. Mathematical analysis of the nucleotide sequences of Protobothrops γPLI-B cDNAs revealed that the particular loops in the three-finger motifs diversified by accelerated evolution. Such evolutionary features should have made serum PLIs acquire their respective inhibitory activities to adapt to venom PLA₂ isozymes.     

Short and Long Form γ2 Subunits of the GABAA/Benzodiazepine Receptors

Abstract: Three novel antisera to the γ₂ subunit of the γ-aminobutyric acid_A (GABA_A) receptor/benzodiazepine receptor (GABA_AR/BZDR) complex have been made. Anti-γ_2S and anti-γ_2L are specific antibodies to synthetic peptides that recognize the γ_2S (short) and γ_2L (long) forms, respectively, of the γ₂ subunit. An antibody (anti-γ₂IL2) to staphylococcal protein A fusion protein of the large intracellular loop (γ₂IL) located between the putative transmembrane segments M3 and M4 of γ_2S recognizes both γ_2S and γ_2L subunits. The antibodies immunoprecipitated both the solubilized and affinity-purified GABA_AR/BZDR from rat and bovine brain. Immunoblots with membranes from rat brain cerebral cortex as well as with affinity-purified receptor from bovine cortex show that anti-γ_2S and anti-γ_2L recognize peptides of 45,000 and 47,000 M_r, respectively. Immunoprecipitation experiments indicate that γ_2S is more prevalent in hippocampus, whereas γ_2L is more abundant in cerebellum. Intermediate values for each form are found in the cerebral cortex. The results suggest that in the rat brain there is a considerable amount of colocalization of γ_2S and γ_2L in the same receptor complex. In the cerebral cortex, 15% of the BZDRs contain both γ_2S and γ_2L subunits and 41–48% of the γ_2L subunit coexists with γ_2S in the same receptor complex. In cerebellum, in 27% of the clonazepam-sensitive and 39% of the clonazepam-insensitive BZDRs the γ_2S and γ_2L coexist in the same receptor complex. The latter are presumably localized in granule cells and also contain α₆. In addition, almost all (93%) the clonazepam-insensitive BZDRs that contain γ_2L also contain a γ_2S subunit in the same receptor complex. The most likely interpretation of the results is that there is an important population of granule cell receptors that contain α₆, γ_2S, and γ_2L coexisting in the same receptor complex. Nevertheless, 31% of the cerebellar receptors that contain α₆ subunit(s) have neither γ_2S nor γ_2L subunits. There are also species differences with respect to the relative abundance of γ_2S and γ_2L. These results might be relevant for understanding the molecular mechanisms underlying some of the GABA_AR/BZDR-mediated effects of ethanol intoxication involving cerebellar granule cells.     

Sequence of interrupted and uninterrupted mRNAs and cloned DNA coding for the two overlapping nonstructural proteins of influenza virus

We have obtained the complete sequence of cloned full-length DNA (NS DNA) derived from influenza virus gene 8, which codes for two unique polypeptides, NS₁ and NS₂, and the sequence of the NS₂ mRNA. Previously we showed that the mRNA for NS₁ (～860 nucleotides) is colinear with the viral RNA and maps from 0.05?0.95 units of the cloned NS DNA, and the body of the NS₂ mRNA (～340 nucleotides) maps from 0.59?0.95 units, suggesting that the two mRNAs are 3′ co-terminal and share the same poly(A) addition site. Sequencing studies have shown that the NS₂ mRNA contains an interrupted sequence of 473 nucleotides. The nucleotide sequences at the junctions of the interrupted segment are similar to those of the consensus sequences at the splicing sites of intervening regions in eucaryotic mRNAs. The first ～56 virus-specific nucleotides at the 5′ end of the NS₂ mRNA are the same nucleotides as are found at the 5′ end of the NS₁ mRNA, and this leader sequence of the NS₂ mRNA contains the initiation codon for protein synthesis and coding information for nine amino acids which would be common to NS₁ and NS₂. In addition, both mRNAs contain 10–20 heterogeneous nonviral nucleotides at their 5′ ends. The ～340 nucleotide body region of the NS₂ mRNA can be translated in the +1 reading frame, and the sequence indicates that NS₁ and NS₂ overlap by 70 amino acids that are translated from different reading frames.     

Functions of milk protein gene 5′ flanking regions on human growth hormone gene

Fragments containing 5′ flanking regions of four bovine milk protein genes—alpha lactalbumin (bαLA), alpha S₁ casein (bαS₁CN), beta casein (bβCN), kappa casein (b_kCN)—and mouse whey acidic protein (mWAP) gene were prepared by PCR and ligated to human growth hormone (hGH) gene. These recombinant DNAs were microinjected into rat embryos to produce transgenic rats, and the functions of the 5′ regions to direct secretion of hGH in the milk were tested. Although milk was obtained only in 5 of 19 mWAP/hGH rat lines, more than two-thirds of the rats carrying the other four DNAs produced milk. More than 80% of the lactated rats carrying bαLA/, bβCN/, and mWAP/hGH, and 33% of the laclated bαS₁CN/hGH rats secreted detectable amounts of hGH (> 0.05 μg/ml) in the milk. In some rats, the hGH concentrations in the milk were comparable to or more than that of the corresponding milk protein in bovine milk. The ranges of hGH concentrations in the milk of bαLA/, bβCN/, bαS₁CN/, and mWAP/hGH rats were 1.13–4,360 μg/ml, 0.11–10,900 μg/ml, 86.8–6,480 μg/ml, and 6.87–151 μg/ml, respectively. HGH was also detected in the sera of these rats, and some abnormalities of growth and reproduction were observed. All but one virgin mWAP/hGH rat secreted up to 0.0722 μg/ml of hGH in the serum, and more than half of them showed abnormal fat accumulations at their abdomen. None of the bαCN/hGH rats secreted detectable amount of hGH into their milk, whereas 8 of the 11 lines secreted hGH into their sera. For the production of hGH in transgenic rat milk, the 5′ region of bαS₁CN was shown most suitable, because the bαS₁CN/hGH rat secreted > 6,000 μg/ml of hGH into the milk and could be reproduced. © 1994 Wiley-Liss, Inc.