Sequence variation at theSec-1 locus of rye,Secale cereale (Poaceae)

This paper describes the structure of a 9.2-kb repeat unit of DNA, which represents one-secalin gene and spacer sequence located at theSec-1 locus on the short arm of chromosome 1 of rye. The gene units at theSec-1 locus comprise 1.1 kb representing the gene and 8.1 kb of spacer sequence separating the genes. A sequence comparison of nine genes and their promoter regions from theSec-1 locus, reveals that there is greater variation within the coding sequence than there is within the promoter regions. The gene sequence variation is discussed in terms of the size variation seen for the-secalin proteins in rye species. The results include a comparison of promoter sequences from members of the Triticeae to examine the degree of conservation between other seed storage protein genes.     

Fungal catabolic gene regulation: Molecular genetic analysis of theamdS gene ofAspergillus nidulans

Aspergillus nidulans is an excellent experimental organism for the study of gene regulation. Genetic and molecular analyses oftrans-acting andcis-acting mutations have revealed a complex pattern of regulation involving multiple independent controls. Expression of theamdS gene is regulated by thefacB andamdA genes which encode positively acting regulatory proteins mediating a major and a minor form of acetate induction respectively. The product of theamdR gene mediates omega amino acid induction ofamdS. The binding sites for each of these proteins have been localised throughamdS cis-acting mutations which specifically affect the interaction with the regulatory protein. The global controls of nitrogen metabolite repression and carbon catabolite repression regulate the expression of many catabolic genes, includingamdS. Nitrogen control is exerted through the positively actingareA gene product and carbon control is dependent on thecreA gene product. Each of the characterized regulatory genes encodes a DNA-binding protein which recognises particular sequences in theamdS promoter to activate or repress gene expression. In addition, there is evidence for other genetically uncharacterised proteins, including a CCAAT-binding complex, which interact with the 5 region of theamdS gene.     

Evolutionary changes ofα-crystallin and the phylogeny of mammalian orders

Summary The sequences of the A chains of the eye lens protein-crystallin from seventeen mammalian species were compared. They showed a generally slow rate of evolution, but with marked variations in different lineages. Most substitutions have occurred in the C-terminal part of the chain, which probably forms part of the surface of the-crystallin aggregate. The ancestral sequence method of Dayhoff revealed interesting indications about the phylogenetic relationships between the eleven mammalian orders that were represented by the investigated species. Most evident was the divergence of marsupial and placental orders. A notable resemblance between the hyrax and elephant sequences was observed, setting them apart from the ungulates, including whale. Primates, rodents, lagomorphs, insectivores and tupaiids seem to derive from a common stem group. These phylogenetic inferences are discussed in relation to current palaeontological and taxonomical opinions, and compared to evidence from other protein sequence data.     

Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants

A set of plasmids has been constructed utilizing the promoter, 5 untranslated exon, and first intron of the maize ubiquitin (Ubi-1) gene to drive expression of protein coding sequences of choice. Plasmids containing chimaeric genes for ubiquitin-luciferase (Ubi-Luc), ubiquitin--glucuronidase (Ubi-GUS), and ubiquitin-phosphinothricin acetyl transferase (Ubi-bar) have been generated, as well as a construct containing chimaeric genes for bothUbi-GUS andUbi-bar in a single plasmid. Another construct was generated to allow cloning of protein coding sequences of choice onBam HI andBam HI-compatible restriction fragments downstream of theUbi-1 gene fragment. Because theUbi-1 promotor has been shown to be highly active in monocots, these constructs may be useful for generating high-level gene expression of selectable markers to facilitate efficient transformation of monocots, to drive expression of reference reporter genes in studies of gene expression, and to provide expression of biotechnologically important protein products in transgenic plants.     

Genomic organization of a mouse MHC class II region including theH2-M andLmp2 loci

The region encompassing theMa, Mb1, Mb2, andLmp2 genes of the mouse class II major histocompatibility complex (MHC) was sequenced. Since this region contains clusters of genes required for efficient class I and class II antigen presentation, it was interesting to search for putative additional genes in the 21 kilobase gap between theMb1 andLmp2 genes. Computer predictions of coding regions and CpG islands, exon trapping experiments, and cross-species comparison with the corresponding human sequence indicate that no additional functional gene is present in that stretch. However, computer analysis revealed the possible existence of an alternative 3 exon forMb1. Except for the fact that the mouse MHC contains twoMb genes, the genomic organization of theH2-M loci was found to be almost identical to the organization of the humanHLA-DM genes. The promoter regions of theMa andMb genes also resemble classical class II promoters, containing typical S, X, and Y boxes. Like the human genes, the threeH2-M genes displayed very limited polymorphism when we compared the cDNA sequences from six haplotypes. Finally, comparison ofDMB withMb1 andMb2, both at the genomic level and in their coding regions, suggests that theMb gene was recently duplicated, probably only in certain rodents.     

Visualization and ablation of phenylethanolamineN-methyltransferase producing cells in transgenic mice

We cloned and sequenced the mouse phenylethanolamineN-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5 flanking sequences from the cloned mouse PNMT gene can direct expression of theEscherichia coli -galactosidase (lacZ) gene to predicted regions of the adrenal, eye can direct in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT_{8 kb}-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.     

Sequence evolution of theGpdh gene in theDrosophila virilis species group

The nucleotide sequence of theGpdh gene from six taxa,D. virilis, D. lummei, D. novamexicana, D. a. americana, D. a. texana andD. ezoana, belonging to thevirilis species group was determined to examine details of evolutionary change in the structure of theGpdh gene. TheGpdh gene is comprised of one 5 non-translated region, eight exons, seven introns and three 3 non-translated regions. Exon/intron organization was identical in all the species examined, but different from that of mammals. Interspecific nucleotide divergence in the entireGpdh gene followed the common pattern: it was low in the exon, high in the intron and intermediate in the non-translated regions. The degree of nucleotide divergence differed within these regions, suggesting that selection exerts constraints differentially on nucleotide change of theGpdh gene. A phylogenetic tree of thevirilis phylad constructed from nucleotide variation of total sequence was consistent with those obtained from other data.Nucleotide sequences for theGpdh gene ofD. lummei, D. novamexicana, D. a. americana, D. a. texana andD. ezoana have been submitted to GenBank with accession numbers D50087, D50088, D50089, D50090 and D50091.     

<Emphasis Type="Italic"> Fugu rubripes</Emphasis> possesses genes for the entire set of the ITAM-bearing transmembrane signal subunits

The transmembrane signaling subunits (TSSs) bearing the immunoreceptor tyrosine-based activation motif (ITAM) play a crucial role in triggering the effector functions of mammalian leukocytes. The involvement in key immune reactions and obvious extension through duplication events make TSSs valuable markers of the evolution of the immune system. We surveyed the genomic sequences of the teleostean fish Fugu rubripes for the presence of genes encoding these accessory molecules. Automatic gene prediction was not efficient because of the poor ability of the programs used to recognize the short exons encoding the intracellular regions of TSSs. However, the unique compactness of the Fugu genome and the conservation of the exon/intron arrangements of the TSS genes facilitated their recognition by visual inspection of the candidate genomic sequences. Evidence for the presence of the CD3, CD3/, CD79a, CD79b, TCR, FcR, DAP12 and DAP10 genes in the Fugu genome was obtained. Furthermore, conserved synteny for the short regions including the TSS genes was revealed by comparison of the Fugu and human genomes. The data demonstrate that the set of TSSs arose before the teleost–tetrapod split and provide a starting point for experimental investigation of the molecular evolution of the leukocyte-activating receptor complexes from fish species to mammals.Abbreviations TSS Transmembrane signal subunit - ITAM Immunoreceptor tyrosine-based activation motif     

TheAdh genomic region ofDrosophila ambigua: Evolutionary trends in different species

Summary The study of individual genes is essential to a comprehensive understanding of genome evolution. The wealth of information on alcohol dehydrogenase (Adh) inDrosophila makes this gene particularly suitable for such analysis. We have characterized more than 4 kb of the genomicAdh region inDrosophila ambigua and compared this region toDrosophila mauritiana andDrosophila pseudoobscura. The presence of two genes,Adh and 3ORF (open reading frame), has been confirmed and some of their essential features have been inferred from primary structural analysis. Inter- and intraspecific comparisons have led us to support that both genes may have diverged from an ancient precursor. They appear to be evolving independently, and show a species-specific pattern. TheAdh in theobscura group species lacks amino acids three and four when compared to the species of themelanogaster group and has accumulated most of its amino acid replacements in the third exon. Neither characteristic is observed when any other group species are compared, which suggests that these may be particular features of the evolution of theobscura group. The 3ORF is highly conserved among the three species analyzed, although variability in the length of the third exon and the nucleotide substitution rate, which is much higher than inAdh, are worth noting. According to our data, both mutation/fixation rates and the distribution of mutations vary over time, which makes it difficult to predict the evolutionary dynamics of specific genome regions.     

The γ-crystallin gene families: Sequence and evolutionary patterns

Summary The -crystallin proteins consist of two topologically equivalent domains, each built up out of two similar motifs. They are encoded by a gene family, which already contained five members before the divergence of rodents and primates. A further gene duplication took place in each lineage. To analyze the pattern of evolution within this gene family, the coding sequences of six human genes, six rat genes, and four mouse genes were compared. Between species, a uniform rate of evolution of all regions of the protein is seen. The ratio of synonymous to nonsynonymous substitution in the human/rat or human/mouse comparison is much lower than the ratio when rat and mouse are compared indicating that the -crystallin proteins are better conserved in the rodent lineage. Within species, the regions encoding the two external motifs I and III of the protein show a greater extent of nonsynonymous substitution than the regions encoding the two internal protein motifs II and IV. The low extent of synonymous substitution between the second exons (encoding motifs I and II) of the rat -crystallin genes suggests the frequent occurrence of gene conversion. In contrast, a high extent of synonymous substitution is found in exon 3 (encoding motifs III and IV) of the rat genes. The same phenomenon is seen within the human gene family. The frequencies of occurrence of the various dinucleotides deviate less from those predicted from the frequencies of occurrence of each individual nucleotide in the second exons than in the third exons. The sequences of the third exons are significantly depleted in CpG, ApA, and GpT and enriched in CpT and GpA.     

Molecular evolutionary analysis of theywvz/7B globin gene cluster of the insectChironomus thummi

We report the sequence of 8.1 kb of DNA containing the 3 end of one and seven other complete intronless globin genes from theywvz/7B locus of the dipteranChironomus thummi thummi. One of these (cttv) appears to be a pseudogene by virtue of a premature termination codon, whereas the others encode apparently functional globin polypeptides. Taken together with previously published data, theC. th. thummi ywvz/7B locus codes for at least 11 globins, five of which differ from one another by no more than two amino acids. In contrast, only nine globin genes are found in a comparable genomic clone isolated fromC. th. piger. As indicated by sequence alignment, this difference in copy number can be attributed to a loss of one gene (fusion of globin genes 7B8 and 7B10) in thepiger lines, coupled with a gain (globin gene 7139) in thethummi lineage. Comparisons between thethummi andpiger sequences showed thatywvz/713 intergenic regions have maintained a level of 91 % similarity since thethummi/piger divergence: most differences are simply due to single base substitutions or insertion/deletion events in either thethummi or thepiger DNA, but three instances of partially overlapping deletions were also detected. A phylogenetic analysis ofywvz/713 gene products was conducted, from which a plausible reconstruction of the evolutionary history of the locus was obtained. In addition, alignment of globin 7B amino acid sequences suggested that globin genes 7B2 and 7B3 (reported at the protein and cDNA level, respectively, but not contained on theC. th. thummi orC. th. piger genomic clones) are possibly chimeric genes. Given the trend toward expansion of theC. thummi globin gene family in general and of the globin 7B subfamily in particular, we propose that increased copy number of these genes has been positively selected as a mechanism to achieve a high Hb concentration in the larval hemolymph. Correspondence to: G. Bergtrom     

The physical map ofMus musculus chromosome 11 reveals evolutionary relationship with different syntenic groups of genes inHomo sapiens

Summary The physical localization of sequences homologous to three cloned genes was determined by in situ hybridization to metaphase chromosomes. Previous work had assigned the skeletal myosin heavy chain gene cluster (Myh), the functional locus for the cellular tumor antigen p53 (Trp53-1), and the cellular homologue of the viral erb-B oncogene (Erbb) toMus musculus chromosome 11 (MMU11). Our results provide regional assignments ofMyh andTrp53-1 to chromosome bands B2C, and ofErbb to bands A1A4. Taken together with in situ mapping of three other loci on MMU 11 (Hox-2 homeobox-containing gene cluster, theSparc protein, and theColla-1 collagen gene), which have been reported elsewhere, these data allowed us to construct a physical map of MMU11 and to compare it with the linkage map of this chromosome. The map positions of the homologous genes on human chromosomes suggest evolutionary relationships of distinct regions of MMU11 with six different human chromosome arms: 1p, 5q, 7p, 16p, 17p, and 17q. The delineation of conserved chromosome regions has important implications for the understanding of karyotype evolution in mammalian species and for the development of animal models of human genetic diseases.     

Considerable haplotypic diversity in the RT1-CE class I gene region of the rat major histocompatibility complex

The major histocompatibility complex (MHC) class I region extending between the Bat1 and Pou5f1 genes shows considerable genomic plasticity in mouse and rhesus macaque but not in human haplotypes. In the rat, this region is known as the RT1-CE region. The recently published rat MHC sequence gave rise to a complete set of class I gene sequences in a single MHC haplotype, namely the RT1ⁿ haplotype of the widely used BN inbred strain. To study the degree of genetic diversity, we compared the RT1-CE region-derived class I genes of the RT1ⁿ haplotype with class I sequences of other rat haplotypes. By using phylogenetic tree analyses, we obtained evidence for extensive presence and absence polymorphisms of single loci and even small subfamilies of class I genes in the rat. Alleles of RT1-CE region class I genes could also be identified, but the rate of allelic nucleotide substitutions appeared rather low, indicating that the diversity in the RT1-CE region is mainly based on genomic plasticity.     

Partial amino acid sequences of mouse transplantation antigens

The partial N-terminal amino acid sequences of the K and D gene products from theH-2 ^q andH-2 ^s haplotypes are presented. These data in conjunction with data already published demonstrate striking homology relationships among the transplantation antigens of mouse and other species. Moreover, these new data support the presence of certain sequence patterns noted in earlier sequence studies (e. g. no Kness or Dness, species-associated residues, and complex allotypes). These patterns place interesting constraints on the genetic organization and evolutionary history of the genes encoding the transplantation antigens which are discussed in this report.Abbreviations used in this paper HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC High performance liquid chromatograph(y) - MEM Minimum essential medium - PTH Phenylthiohydantoin - SDS Sodium dodecyl sulfate     

Multigenic regulation of the primary immune response to ovalbumin in mice

At least four genes regulate the primary immune response to ovalbumin in mice. The ability to be sensitized to transfer delayed type hypersensitivity to ovalbumin is controlled by two genes. One gene,OVA-, is linked to theH-2 complex and maps to the left ofI-E. The linkage of the other gene,OVA-Bg1, has not been determined, but it segregates independently of theLy M locus, of the heavy chain allotype genes and of certain genes controlling coat color. At least two genes regulate the ability to respond with a primary antiovalbumin antibody response. One gene,OVA-, is linked to theH-2 complex and maps to the right ofI-E. Discordance of the minimum dose of antigen needed to elicit delayed type hypersensitivity response and antibody suggests that non-H-2 gene(s) regulating the primary antibody response are different fromOVA-Bg1.Abbreviations used in this paper BSA bovine serum albumin - DTH delayed type hypersensitivity - H-2 major histocompatibility complex of mouse - Ir gene — immune response gene - OVA ovalbumin - SRBC sheep red blood cells     

Definition of microsatellite size variants forTnfa andHsp70 in autoimmune and nonautoimmune mouse strains

We have cloned and sequenced the upstream regulatory region of tumor necrosis factor (Tnfa) gene in 12 different mouse strains and identified an allelic polymorphism in the upstream regulatory region of the mouseTnfa gene. TheTNF allele found in the NZW strain is distinct from those of all otherH-2 haplotypes, supporting our previous suggestion that this allele may be associated with a regulatory or structural defect. In addition, simple tandem repeat sequences (microsatellites) within the promoter region of theTnfa gene and the 3 untranslated region of one of the members of the HSP70 family (Hsp68c clone) were utilized as genetic markers. Ten TNF size variants and twelve HSP70 variants were identified in over forty mouse strains. Using these markers in a set of congenic mice, we mapped this member of the HSP70 family to the central portion of theH-2 complex, centromeric to theTnfa gene. The NOD and NZW strains carry uniqueHSP70 alleles based on the variability in the length of this marker. These findings raise the possibility that this protein may play a role in the association of the major histocompatibility complex with these autoimmune diseases.     

Drosophila mitochondrial DNA: Conserved sequences in the A+T-rich region and supporting evidence for a secondary structure model of the small ribosomal RNA

Summary The sequence of a segment of theDrosophila virilis mitochondrial DNA (mtDNA) molecule that contains the A+T-rich region, the small rRNA gene, the tRNA^f-met, tRNA^gln, and tRNA^ile genes, and portions of the ND2 and tRNA^val genes is presented and compared with the corresponding segment of theD. yakuba mtDNA molecule. The A+T-rich regions ofD. virilis andD. yakuba contain two correspondingly located sequences of 49 and 276/274 nucleotides that appear to have been conserved during evolution. In each species the replication origin of the mtDNA molecule is calculated to lie within a region that overlaps the larger conserved sequence, and within this overlap is found a potential hairpin structure. Substitutions between the larger conserved sequences of the A+T-rich regions, the small mt-rRNA genes, and the ND2 genes are biased in favor of transversions, 71–97% of which are AT changes. There is a 13.8 times higher frequency of nucleotide differences between the 5 halves than between the 3 halves of theD. virilis andD. yakuba small mt-rRNA genes. Considerations of the effects of observed substitutions and deletion/insertions on possible nucleotide pairing within the small mt-rRNA genes ofD. virilis andD. yakuba strongly support the secondary structure model for theDrosophila small mt-rRNA that we previously proposed.     

Evolutionary analysis ofα andβ hemoglobin genes by reh theory under the assumption of the equiprobability of genetic events

Summary It is shown how REH theory in conjunction with mRNA or gene sequence data can be used to obtain estimates of the fixation intensity, the number of varions, and the total mutations fixed between homologous pairs of nucleic acids. These estimates are more accurate than those that can be derived from amino acid sequence data. The method is illustrated for and hemoglobin genes and these improved estimates are compared with those made from the amino acid sequences for which those genes code. Significant differences are found between the estimates made by these two methods. For the hemoglobin gene sequences examined here, the fixation intensity is some-what less than the protein data had suggested, and the number of rations is considerably greater. Depending on the gene sequences examined, between 62 and 83% of the codons appear able to fix mutations during the divergences considered. This reflects the constraints of natural selection on acceptable mutations. The total number of base replacements separating the genes for human, mouse, and rabbit hemoglobin varies from 61 to 105 depending on the pair examined. Rabbit and hemoglobin are separated by at least 290 fixed mutations. For such distantly related sequences estimates made from protein and mRNA data differ less, reflecting the higher quality of information from the many observed changes in primary structure. The effects of nonrandom gene structure on these evolutionary estimates and the fact that various genetic events are not equiprobable are discussed.     

Independent gene duplications,not concerted evolution,explain relationships among class I MHC genes of murine rodents

It has been claimed that class I MHC loci are homogenized within species by frequent events of interlocus genetic exchange (concerted evolution). Evidence for this process includes the fact that certain rat class I loci (including RT1.A) located centromeric to class II and class III are more similar to each other than to the mouse K locus (also centromeric to class II/class III). However, a phylogenetic analysis showed that the rat RT1.A locus is in fact orthologous to the mouse K1 pseudogene (also centromeric to class II/class III). Thus, two independent events of translocation of genes centromeric to class II/class III have occurred in the history of the murine rodents, at least one of which (involving the ancestor of RT1.A and K1) occurred prior to the divergence of rat and mouse. It was also found that the rat nonclassical class I gene RT.BM1 is orthologous to the mouse nonclassical gene 37 ^d. These results argue that intelocus genetic exchange does not occur at a rate sufficient to cause within-species homogenization of class I MHC loci.     

Molecular Cloning and Chromosomal Localization of the MouseGpr37Gene Encoding an Orphan G-Protein-Coupled Peptide Receptor Expressed in Brain and Testis

We report the cloning of the mouse ortholog of the humanGPR37gene, which encodes an orphan G-protein-coupled receptor highly expressed in brain tissues and homologous to neuropeptide-specific receptors ([20],Genomics 45:68–77;[45],Biochem. Biophys. Res. Commun. 233:559–567). The genomic organization of theGPR37gene is conserved in both mouse and human species with a single intron interrupting the receptor-coding sequence within the presumed third transmembrane domain. Comparative genetic mapping of theGPR37gene showed that it maps to a conserved chromosomal segment on proximal mouse chromosome 6 and human chromosome 7q31. The mouseGpr37gene contains an open reading frame coding for a 600-amino-acid protein 83% identical to the humanGPR37gene product. The predicted mouse GPR37 protein contains seven putative hydrophobic transmembrane domains, as well as a long (249 amino acid residues), arginine- and proline-rich amino-terminal extracellular domain, which is also a distinctive feature of the human GPR37 receptor. Northern blot analysis of mouse tissues withGpr37-specific probes revealed a main 3.8-kb mRNA and a much less abundant 8-kb mRNA, both expressed in the brain. A 3-kb mRNA is also expressed in the testis. Both the mouse and the humanGPR37genes may belong to a class of highly conserved mammalian genes encoding a novel type of G-protein-coupled receptor predominantly expressed in the brain.