The evolution of protamine P1 genes in dasyurid marsupials

We report the complete DNA sequences of the protamine P1 gene and flanking regions for 13 species of the marsupial family Dasyuridae. The structure of the protamine locus is conserved in dasyurids and consists of two exons (of lengths 142–151 and 47 bp) separated by an intron (208–240 bp). A key feature of the dasyund intron is a 38–40 by duplication found in all species examined to date. This duplication apparently predates the radiation of modern dasyurid lineages and may be homologous to a similar feature in the marsupial mole (Notoryctes). Sequences from a species of Planigale demonstrate that this genus is unique among marsupials in possessing cysteine residues in its protamine P1 molecules. Cysteines may provide enhanced chemical stability for condensed sperm nuclei, a physiological feature that would converge on the common eutherian pattern. Phylogenetic analysis of the protamine genes yields a tree that is largely congruent with previous molecular systematic studies in two areas: (1) There are three main dasyurid lineages corresponding to the Sminthopsinae, Dasyurinae, and Phascogalinae; (2) Dasyurinae and Phascogalinae are sister groups. This study is the first estimate of dasyurid relationships based on a nuclear DNA sequence. Correspondence to: J.D. Retief     

Variability and inheritance of histone genes H3 and H4 in Vicia faba

Summary We have compared copy numbers and blothybridization patterns of histone genes (H3 plus H4) between and within individuals of broad bean (Vicia faba). Copy number differences among individuals in the population of 200 individuals were as great as 27 fold, and as much as 3.2 fold among separate leaves of the same plant. Among F₂ progeny from genetic crosses, up to a 5.4-fold range was seen (mean=3.5 fold), and among F₁ progeny of self-pollinated plants, up to a 5.9-fold range was observed (mean=2.3 fold). Histone gene blot-hybridization patterns for EcoRI and HindIII were also variable among individuals and indicated that the genes are probably clustered in only a few chromosomal loci. The degree of variation in histone gene copy number per haploid genome (2–55 copies, or 27 fold) was similar to that found previously for ribosomal RNA genes (230–22000, or 95 fold) of V. faba. However, the two gene families change independently, since individuals with a high or low copy number for one gene can have either a high or low copy number for the other. The mechanisms(s) for rapid gene copy number change may be similar for these gene families.     

Assignment of the βB1 Crystallin Gene (CRYBB1) to Human Chromosome 22 and Mouse Chromosome 5

By using primers complementary to the rat βB1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences.CRYBB1was assigned to the group 5 region in 22q11.2–q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products ofCRYBB1were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other β crystallin genes (βB2(−1), βB3, and βA4) have previously been mapped to the same regions in human and mouse. We demonstrate that the βB1 and βA4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major β crystallins genes that are expressed in the bovine lens.     

Larger rearranged mitochondrial genomes in Dekkera/Brettanomyces yeasts are more closely related than smaller genomes with a conserved gene order

Summary Mitochondrial genomes from yeasts in the Dekkera/Brettanomyces/Eeniella group vary in size from 28 to 101 kb. Mapping of genes has shown that the three smallest genomes, of 28–42 kb, have the same gene order, whereas the three larger mitochondrial DNAs of 57–101 kb are rearranged relative to the smaller molecules and between themselves. To examine the relationships between these genomes, a phylogenetic tree has been constructed by sequence comparison of the mitochondrialencoded cytochrome oxidase subunit gene (COX2) from the six species. Contrary to expectation, the tree shows that the larger rearranged genomes are more closely related than the smaller mtDNAs. This result indicates that the gene order of the smaller mtDNAs (28–42 kb) is ancestral and that larger mtDNA molecules (57–101 kb) are more prone to rearrangement than smaller forms.Offprint requests to: G.D. Clark-Walker     

Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6–8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (<5 μg/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent K_m of 60 μM and V_max of 20 μmol·min⁻¹·mg⁻¹, corresponding values with mixed histones were 12 μM and 1.2 μmol·min⁻¹·mg⁻¹. With both protein substrates the apparent K_m for ATP was 11 μM. Concentrations of Mg²⁺ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7–2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated ³²P_i from [γ-³²P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.     

Evolution of protamine P1 genes in primates

Protamine P1 genes have been sequenced by PCR amplification and direct DNA sequencing from 9 primates representing 5 major families, Cebidae (new world monkeys), Cercopithecidae (old world monkeys), Hylobatidae (gibbons), Pongidae (gorilla, orangutan, and chimpanzee), and Hominidae (human). In this recently diverged group of primates these genes are clearly orthologous but very variable, both at the DNA level and in their expressed amino acid sequences. The rate of variation amongst the protamine Pls indicates that they are amongst the most rapidly diverging polypeptides studied. However, some regions are conserved both in primates and generally in other placental mammals. These are the 13 N-terminal residues (including a region of alternating serine and arginine residues (the motif SRSR, res. 10–13) susceptible to Ser phosphorylation), a tract of six Arg residues (res. 24–29) in the center of the molecule, and a six-residue region (RCCRRR, res. 39–44), consisting of a pair of cysteines flanked by arginines. Detailed consideration of nearest neighbor matrices and trees based on maximum parsimony indicates that PI genes from humans, gorillas, and chimpanzees are very similar. The amino acid and nucleotide differences between humans and gorillas. are fewer than those between humans and chimpanzees. This finding is at variance with data from DNA-DNA hybridization and extensive globin and mitochondrial DNA sequences which place human and chimpanzee as closest relatives in the super family, Hominoidea. This may be related to the fact that protamine Pls are expressed in germ line rather than somatic cells. In contrast to the variability of the exon regions of the protamine P1 genes, the sequence of the single intron is highly conserved.     

Low- and intermediate-copy-number cloning vectors based on thePseudomonas plasmid pVS1

The cloning vector pME290 (6.8 kb), which is derived from thePseudomonas plasmid pVS1 and has about 7 copies, was mutagenized in vitro to provide derivatives with altered copy numbers. Thus, pME292 (about 1–3 copies) and pME294 (about 15–20 copies) were isolated. These vectors were used in the characterization of theP. aeruginosa argF gene encoding ornithine carbamoyltransferase.     

Highly Variable Polymorphism of the α-Amylase Gene Family in <Emphasis Type="Italic">Litopenaeus</Emphasis><Emphasis Type="Italic">vannamei</Emphasis> (Crustacea Decapoda)

-Amylase from the tropical shrimp Litopenaeus vannamei presents a high degree of polymorphism and at least eight different electromorphs are detected by electrophoresis. Based on nucleotide sequences, three cDNAs have been previously characterized. In this paper we report on the organization and the evolution of corresponding -amylase genes, determined after PCR amplification. Three AMY genes have been characterized, spanning over 3.3 kb and encoding mature proteins of 495 amino acids (aa), which are all expressed in the digestive gland. The existence of nine short introns, ranging from 86 to 454 bp, located at the same positions for each of the different genes, and presenting no similarity between them, is reported. Between 11 and 15% of changes are observed in the coding aa sequences of genes II and III compared to the gene I sequence respectively. One 5 putative promoter sequence has been sequenced and shows no classical TATA box upstream to the coding sequence. Based on the intron size difference, a single PCR (producing the S–R fragments) allows the separation of a partial gene I (750 bp), corresponding to cDNA 20, from the others (650–680 bp). Sequencing different S–R PCR fragments from one shrimp shows at least eight different haplotypes. A complex microsatellite repeat is present in intron 6 of gene II. Using size and sequence differences in this repeated portion, it is possible to characterize two gene subfamilies (IIa and IIb) encoding previously described cDNAs 28 and 37, respectively. For the gene II family, two to four alleles are present in one shrimp corresponding to these two genes. Within the Panama natural population, 35 different alleles are shown at this locus. Regarding -amylase gene structure in the shrimp, many recombinants are present from a set of individuals and constitute an important mechanism of evolution of -amylase function. Accession numbers: AJ132379, L. vannamei -amylase gene I; AJ133526, gene II; AJ133119, gene III     

Morphogenesis of the tail fiber of bacteriophage T 4 . V. In vitro complementation between the gene 36 product and the genes 37-38 directed component

Summary An in vitro complementation was observed between the gene 36 product and the genes 37–38 directed component of the tail fiber of bacteriophage T₄. A possible role of the gene 36 product as well as the reconstitution process in the complementation were briefly discussed.Biozentrum, University of Basel, CH-4056 Basel, Swiss. 1 The following defectives in T₄ fiber genes were used: single defective mutants; amE1 (gene 36^–), amN52 (gene 37^–) and amB262 (gene 38^–); double defective mutants; amN52:B262 (genes 37^–38^–), amA455: N52 (genes 34^–37^–) and amB252:N52 (genes 35^–37^–); and triple defective mutants; am A455:B252:N52 (genes 34^–35^–37^–) and amA455:B252:B262 (genes 34^–35^–38^–).     

Chromosomal Assignment of Four Testis-Expressed Mouse Genes from a New Family of Transmembrane Proteins (ADAMs) Involved in Cell–Cell Adhesion and Fusion

A new gene family of multidomain membrane proteins (ADAMs) that include isintegrin nd etalloprotease domain comprises an increasing number of identified members. Two members of this family, fertilin α and fertilin β, form a heterodimeric protein that is required for sperm–egg fusion. Most recently, it has been shown that a third family member, meltrin α, is involved in myoblast fusion (Yagami-Hiromasaet al.,1995,Nature377: 652–656). Using restriction fragment length polymorphism analysis of a DNA panel from an interspecific backcross, we have determined the chromosomal locations of four mouse genes of this family that are expressed in testis: fertilin α, fertilin β, ADAM 4, and ADAM 5. These genes have been given the locus symbolsFtna(fertilin α),Ftnb(fertilin β),Adam4(ADAM 4), andAdam5(ADAM 5). They were mapped to chromosomes 5, 14, 9, and 8, respectively, revealing a dispersed localization. Human chromosome locations of these genes are predicted on the basis of the mapping results using the information provided by comparative linkage maps. Because all four of these ADAM genes are expressed in testis and fertilin α and β have been found to be important for fertilization, we compared their chromosomal locations with known mouse mutations affecting spermatogenesis and fertility.     

Intergeneric Relationships Among Macropodoidea (Metatheria: Diprotodontia) and The Chronicle of Kangaroo Evolution

The superfamily of kangaroos (Macropodoidea) is comprised of the subfamilies Propleopinae, Hypsiprymnodontinae, Paleopotoroinae, Potoroinae, Bulungamayinae, Balbarinae, Macropodinae, and Sthenurinae. Of these, Hypsiprymnodontinae, Potoroinae, and Macropodinae are extant. Competing phylogenetic hypotheses unite potoroines with either hypsiprymnodontines or macropodines, with most recent workers following a classificatory scheme that recognizes Hypsiprymnodontidae (hypsiprymnodontines) and Macropodidae (macropodines + potoroines). To address phylogenetic relationships among living macropodoids, we analyzed sequences from three mitochondrial genes (12S rRNA, tRNA valine, 16S rRNA) and one nuclear gene (protamine P1). MtDNA and protamine P1 both support a basal split of Hypsiprymnodon from other macropodoids rather than an association of Hypsiprymnodon with potoroines. This suggests that bipedal hopping and a complex stomach evolved once among macropodids. Monophyly of the Macropodinae is supported. Among macropodines, there is support for a Dorcopsis-Dorcopsulus association. Potoroine monophyly is less clear, although among potoroines there is support for an association of Bettongia and Aepyprymnus. Divergence times were estimated using 12S rRNA, tRNA-valine, and 16S rRNA transversions and suggest that kangaroos separated from a possum-like ancestor approximately 38–44 million years ago. Hypsiprymnodon diverged from other macropodoids approximately 34 to 38 million years ago. In agreement with the fossil record, the diversification of potoroines predates the diversification of macropodines. The latter have radiated in association with the development of a more arid climate and emergent grasslands over the Australian continent.     

High-Resolution Linkage Map of Mouse Chromosome 13 in the Vicinity of the Host Resistance LocusLgn1

Natural resistance of inbred mouse strains to infection withLegionella pneumophilais controlled by the expression of a single dominant gene on chromosome 13, designatedLgn1.The genetic difference atLgn1is phenotypically expressed as the presence or absence of intracellular replication ofL. pneumophilain host macrophages. In our effort to identify theLgn1gene by positional cloning, we have generated a high-resolution linkage map of theLgn1chromosomal region. For this, we have carried out extensive segregation analysis in a total of 1270 (A/J × C57BL/6J) × A/J informative backcross mice segregating the resistance allele of C57BL/6J and the susceptibility allele of A/J. Additional segregation analyses were carried out in three preexisting panels of C57BL/6J ×Mus spretusinterspecific backcross mice. A total of 39 DNA markers were mapped within an interval of approximately 30 cM overlapping theLgn1region. Combined pedigree analyses for the 5.4-cM segment overlappingLgn1indicated the locus order and the interlocus distances (in cM):D13Mit128–(1.4)–D13Mit194–(0.1)–D13Mit147–(0.9)–D13Mit36–(0.9)–D13Mit146–(0.2)–Lgn1/D13Mit37–(1.0)–D13Mit70.Additional genetic linkage studies of markers not informative in the A/J × C57BL/6J cross positionedD13Mit30, -72, -195,and-203, D13Gor4, D13Hun35,andMtap5in the immediate vicinity of theLgn1locus. The marker density and resolution of this genetic linkage map should allow the construction of a physical map of the region and the isolation of YAC clones overlapping the gene.     

Drosophila virilis histone gene clusters lacking H1 coding segments

Summary Approximately 30–40% ofDrosophila virilis DNA complementary to clonedDrosophila histone genes is reduced to 3.4-kilobase-pair (kbp) segments by Bgl I or Bgl II digestion. The core histone genes of a 3.4-kbp Bgl II segment cloned in the plasmid pDv3/3.4 have the same order as theD. melanogaster core histone genes in the plasmid cDm500: . Nonetheless, pDv3/3.4 and cDm500 have different histone gene configurations: In pDv3/3.4, the region between the H2B and H3 genes contains 0.35 kbp and cannot encode histone H1; in cDm500, the region contains 2.0 kbp and encodes histone H1. The lack of an H1 gene between the H2B and H3 genes in 30–40% ofD. virilis histone gene clusters suggests that changes in histone gene arrays have occurred during the evolution ofDrosophila. The ancestors of modernDrosophila may have possessed multiple varieties of histone gene clusters, which were subsequently lost differentially in thevirilis andmelanogaster lineages. Alternatively, they may have possessed a single variety, which was rearranged during evolution. The H1 genes ofD. virilis andD. melanogaster did not cross-hybridize in vitro under conditions that maintain stable duplexes between DNAs that are 75% homologous. Consequently,D. virilis H1 genes could not be visualized by hybridization to an H1-specific probe and thus remain unidentified. Our observations suggest that the coding segments in the H1 genes ofD. virilis andD. melanogaster are >25% divergent. Our estimate of sequence divergence in the H1 genes ofD. virilis andD. melanogaster seems high until one considers that the coding sequences of cloned H1 genes from the closely related speciesD. melanogaster andD. simulans are 5% divergent.     

The α-Globin Gene Family of an Australian Marsupial, Macropus eugenii: The Long Evolutionary History of the θ-Globin Gene and Its Functional Status in Mammals

Comparative evolutionary analyses of gene families among divergent lineages can provide information on the order and timing of major gene duplication events and evolution of gene function. Here we investigate the evolutionary history of the α-globin gene family in mammals by isolating and characterizing α-like globin genes from an Australian marsupial, the tammar wallaby, Macropus eugenii. Sequence and phylogenetic analyses indicate that the tammar α-globin family consists of at least four genes including a single adult-expressed gene (α), two embryonic/neonatally expressed genes (ζ and ζ′), and θ-globin, each orthologous to the respective α-, ζ-, and θ-globin genes of eutherian mammals. The results suggest that the θ-globin lineage arose by duplication of an ancestral adult α-globin gene and had already evolved an unusual promoter region, atypical of all known α-globin gene promoters, prior to the divergence of the marsupial and eutherian lineages. Evolutionary analyses, using a maximum likelihood approach, indicate that θ-globin, has evolved under strong selective constraints in both marsupials and the lineage leading to human θ-globin, suggesting a long-term functional status. Overall, our results indicate that at least a four-gene cluster consisting of three α-like and one β-like globin genes linked in the order 5′–ζ–α–θ–ω–3′ existed in the common ancestor of marsupials and eutherians. However, results are inconclusive as to whether the two tammar ζ-globin genes arose by duplication prior to the radiation of the marsupial and eutherian lineages, with maintenance of exon sequences by gene conversion, or more recently within marsupials.Reviewing Editor: Dr. John Oakeshott     

Phylogeny of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Genes in Haloalkaliphilic Obligately Autotrophic Sulfur-Oxidizing Bacteria of the Genus <Emphasis Type="Italic">Thioalkalivibrio</Emphasis>

Fragments of genes of the “green-like” form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of eight species of haloalkaliphilic obligately autotrophic sulfur-oxidizing bacteria of the genus Thioalkalivibrio have been revealed and sequenced using previously developed oligonucleotide primers. The data obtained are used for the construction of phylogenetic trees on the basis of nucleotide sequences of RuBisCO genes and their conceptual translations into amino acid sequences. Comparative analysis of the 16S rRNA and RuBisCO gene trees reveals discrepancies between their topologies. According to a RuBisCO gene analysis, the genus Thioalkalivibrio is not monophyletic, and its inner divergence conforms to the significant morphological differences observed between the species. Presumably, horizontal (interspecies) gene transfer was involved in the evolution of the genus Thioalkalivibrio.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 378–386.Original Russian Text Copyright © 2005 by Tourova, Spiridonova, Berg, Kuznetsov, Sorokin.