Cloning and expression of β-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Summary The -galactosidase gene ofStreptococcus thermophilus was cloned into plasmid vector, pVT100-U, and used to transform a strain ofEscherichia coli andSaccharomyces cerevisiae. Transformants which expressed -galactosidase activity were obtained in bothE. coli andSaccharomyces cerevisiae, the highest activity found in a yeast recombinant. The expression and thermostability of the cloned -galactosidase genes from different plasmid constructions were compared with the streptococcal -galactosidase. The recombinant protein was equivalent to the specific activity and thermostability ofS. thermophilus.     

Plant growth regulatory metabolites from novel actinomycetes

Metabolites from 796 isolates of aerobic actinomycetes were screened for plant growth regulatory properties using an algal bioassay. These included 266 isolates ofStreptomyces, 28 unidentified actinomycetes, and 502 isolates of novel actinomycetes represented by 18 genera. Algal growth inhibition of 30% was observed with 60 isolates, 37 of which belonged to the genusStreptomyces. Among other inhibitors were 8 isolates ofActinomadura, 6 ofActinoplanes, 2 each of the generaThermomonospora, Streptoverticillium, andPromicromonospora, and 3 unidentified. Metabolites from 70 isolates promoted algal growth by 20%. These included 13 isolates ofMicromonospora, 11 ofStreptomyces, 6 ofNocardia, 5 ofActinomadura, and 4 each ofRhodococcus andThermomonospora. Sixteen unidentified isolates; 3 isolates ofPromicromonospora; 2 isolates each ofActinoplanes, Streptosporangium, andOerskovia; and 1 of Thermoactinomyces peptonophilus-like organism andSaccharomonospora viridis also promoted the algal growth by 20%. The plant growth inhibitory properties of 9 actinomycetes and the growth promoting properties of 6 were demonstrable during the secondary screening on higher plants using chemicals extracted from the culture broth. The metabolites fromMicromonospora, Nocardia, Rhodococcus, Streptosporangium, andOerskovia isolates were associated with plant growth promotion only; those fromStreptomyces were most frequently involved with the growth inhibition.This is Michigan Agriculture Experiment Station Journal Article No. 12191.     

Regeneration and Agrobacterium-mediated transformation of Forsythia×intermedia "Spring Glory"

Internode explants ofin vitro plants ofForsythia x intermedia Spring Glory were transformed with thegus andnpt II genes after inoculation with theA. tumefaciens strain EHA 101 harbouring the plasmid pFAJ3000. Shoot organogenesis took place from callused edges of explants. The first transformed buds were detected 4 to 6 weeks after transfer on regeneration medium, containing 25 mg/l kanamycin as selective agent. An average of 1% of explants regenerated transgenic shoots.-glucuronidase assays and culture on kanamycin-containing medium provided the first indication of integration and expression of introduced genes in transformants. Southern blot and polymerase chain reaction amplification analyses gave molecular confirmation of genetic transformation. Transgenic plants were acclimatized in the greenhouse. Enzymatic assays on several organs of mature plants still showed -glucuronidase activity, thus confirming stable integration of T-DNA in the plant genome.Abbreviations BAP 6-benzyl-aminopurine - CaMV Cauliflower Mosaic Virus - GUS andgus -glucuronidase - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog - NOS nopaline synthase - NPT II andnpt II neomycin phosphotransferase II - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - SSC sodium chloride-sodium citrate - X-Gluc 5-bromo-4-cbloro-3-indolyl glucuronide     

Fibrinogen γ chain locus is on chromosome 4 in man

Summary A molecular fibrinogen variant has been detected by two-dimensional electrophoresis of human plasma samples. Fibrinogen is a complex molecule consisting of three different polypeptide chains A, B, and . The presently described variation resides in the -chain, which in the variant is slightly more basic and heavier than the common form of this chain. In a family material it has been shown that the variant is genetically determined, and the segregation pattern shows autosomal codominant inheritance. The family material has been typed in approximately 30 marker systems, and linkage studies have shown close linkage between the -chain locus (FGG) and MNSs. The MNSs loci are known to be located on chromosome 4 in humans and the fibrinogen -chain locus is thus on this chromosome. The MNSs/FGG distance is approximately 8 centimorgans. Supplementing data suggest that FGG is distal to MNSs on the long arm of chromosome 4.     

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.     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

Studies on the fungus flora of three grains in Egypt

In wheat, corn and sorghum grains twenty-six genera and seventy-seven species including sixteen ofAspergillus and twenty-one ofPenicillium were identified.In grain samples adjusted to various moisture levels (up to 18.8 % on a dry-weight basis) and stored for 4 months at 8, 20 and 30 C seed-borne fungi were monthly identified and counted, and the germinability of the grains was tested. In the control samples (moisture content 7.1–8.2 %) temperature had no effect on the fungal counts and on the precentage germination. With the rise of temperature and moisture content the fungal counts markedly increased and the germinability declined.The list of fungi dominant in one or more of the experiments, included eight species ofAspergillus, six ofPenicillium, two ofFusarium and one each ofAlternaria andCurvularia. The order of dominance of these fungi varied according to the type of grain, the length of storage and the treatment.A. niger was the dominant organism in the control samples of the three grains. It could grow successfully at high moisture contents but above 15 %, it was usually overgrown by other fungi. In wheatP. citrinum andA. sydowii, in cornP. citrinum andA. terreus, and in sorghumA. terreus andA. niger were the dominant fungi at moisture contents above 15 % after four months storage at 30 C. When stored at 8 CPenicillium species tended to predominate over other fungi in grains with high moisture contents.In surface-sterilized grains adjusted to 15 % moisture content, inoculated with six dominant fungi separately and incubated at 30 C,A. niger, P. citrinum andP. variabile caused severe deterioration in the three grains;A. ochraceus in wheat only andF. moniliforme in wheat and corn;C. spicifer was slightly injurious to the three grains.     

Sexual compatibility between serotypes ofFilobasidiella neoformans (Cryptococcus neoformans)

Twenty-five of 37Cryptococcus neoformans strains of known serotype produced the basidiomycetousFilobasidiella state either alone or when paired with a strain of compatible mating-type. Sixteen strains were mating-type, four strains were a mating-type, and five strains were self-fertile.F. neoformans serotypes A and D were interfertile with compatible mating-types ofF. bacillispora serotypes B and C.C. neoformans var.gattii was interfertile with compatible mating-types ofF. neoformans andF. bacillispora. F. bacillispora strains, which utilized creatinine andl-malic acid, were interfertile with compatible mating-types ofF. neoformans, which did not utilize creatinine andl-malic acid. The interfertility between serotypes and biotypes eliminates the need for recognizing the names ofC. neoformans var.gattii, C. bacillisporus, andF. bacillispora. It is proposed thatC. neoformans var.gattii andC. bacillisporus be regarded as later, facultative synonyms ofC. neoformans and thatF. bacillispora be regarded as a later, facultative synonym ofF. neoformans.     

Chemosystematics of theRutaceae: Suggestions for a more natural taxonomy and evolutionary interpretation of the family

The chemosystematics ofRutaceae is reviewed on the basis of updated surveys of various secondary metabolites and their biosynthetic derivation. A comparison of these data with the morphological and geographical differentiation clearly shows that the current taxonomic arrangement of the family is to a large extent artificial and needs improvement. Starting from obviously natural groups of genera (or single genera) as basic taxonomic entities a new system with informal tribal names is suggested. In particular, the subfamilyToddalioideae is broken up altogether and its former members are rearranged among several of the 17 provisional tribes within the subfamilyRutoideae s. lat. Phylogenetic progressions can be recognized from parallel changes of morphological characters and biosynthetic pathways to secondary metabolites. As a general trend, a stepwise replacement of benzylisoquinolines by simple and complex anthranilic acid derived alkaloids, and eventually by coumarins and/or limonoids is confirmed. The available data are summarized in a discussion of the possible evolutionary relationships among theRutaceae, with theZanthoxylum- andEvodia-tribes in a central position.     

Genetic mapping of the component chains of Ia antigens

The genes encoding the two polypeptide chains ( and) that comprise the murine Ia antigens were localized within distinct regions of the major histocompatibility complex (MHC). This was accomplished by correlating allelic forms of the and chains with the MHC congenic strains of mice from which they were isolated. Allelic forms of and chains were distinguished by their unique structural markers, such as isoelectric points, amino acid sequences or peptide maps. The results indicate that the structural genes for both the and chains of I-A subregion antigens are located within the K to I-A genetic interval. In contrast, the gene encoding the chain of I-E subregion antigens is located outside of theI-E subregion and within the K to I-B genetic interval. These findings may have important implications for analysis of observations that complementation by twoI-region genes is sometimes required for development of immune responses.     

Teliospores of smut fungi teliospore connections, appendages, and germ pores studied by electron microscopy; phylogenetic discussion of characteristics of teliospores

Summary Special features of teliospores in smut fungi are described, including teliospore connections, appendages, and germ pores. Balls of teliospores in species of many different genera cohere by remnants of hyphal walls, sheaths, and sometimes interlocking ornamentation. Teliospores are connected in pairs in species ofMycosyrinx andGeminago by special local structures. Appendages can be formed locally by persistent material from the sheath (Cintractia, Anthracoidea, Sphacelotheca), thickened parts of the spore wall (e.g.,Georgefischeria, Jamesdicksonia, Rhamphospora, Tolyposporella), or persistent walls of sporogenous hyphae (Rhamphospora, genera of the Tilletia relationship). Species ofGeorgefischeria, Jamesdicksonia, andTolyposporella have teliospore walls composed of more than three layers of different electron density. Germ areas corresponding to thinner parts of the spore wall are known, e.g., for species ofAnthracoidea, Cintractia, andUstilago infecting members of the family Poaceae, while distinct germ pores, one per teliospore, are found in some species ofThecaphora, Tolyposporium, andSporisorium. Teliospores ofMycosyrinx cissi have a germination ring. Characteristics of teliospores are used to discuss the phylogeny of smut fungi. A phylogenetic tree in accordance with teliospore characteristics is compared to those obtained from ultrastructural characteristics of host-parasite interaction, of septal pores, and from sequence data. Aspects of teliospore development help to define taxa at a high systematic level (Entorrhizales, Ustilaginales, Tilletiales/Entylomatales, Microbotryaceae), while details of ornamentation ontogeny delimit groups of genera (e.g., genera related toUstilago on members of the Poaceae andSporisorium, Cintractia andAnthracoidea, Tilletia) or single genera (e.g.,Melanopsichium, Dermatosorus, Mycosyrinx, Doassinga, Rhamphospora). Types of ornamentation (warty, reticulate), middle layers, teliospore balls, and germ pores evolved repeatedly by convergence. The smut teliospore itself probably evolved independently at least twice, or perhaps three (or more) times, in the Microbotryales, in the Entorrhizales, and in a common ancestor of the remainder of the Ustilaginomycetes.Part 166 in the series Studies in Heterobasidiomycetes from the Botanical Institute, University of Tübingen.     

Early expression of two<Emphasis Type="Italic"> TdT</Emphasis> isoforms in the hematopoietic system of the Mexican axolotl

Nontemplate (N)-nucleotide addition by the terminal dideoxynucleotidyl transferase (TdT) at the junctions of rearranging V(D)J gene segments greatly contribute to antigen-receptor diversity. TdT has been identified in several vertebrate species, where it is highly conserved. We report here the isolation of two forms of TdT mRNA in an amphibian, the Mexican axolotl. The isoform TdT1 shares all of the conserved structural motifs required for TdT activity and displays an average of 50–58% similarity at the amino acid level with TdT of other species. The second axolotl TdT variant (TdT2) differs from TdT1 by a 57-amino acid deletion located between amino acids 165–222 of TdT1, including the first helix–hairpin–helix DNA-binding motif. During ontogeny, TdT products are first detected in the head of 6-week-old larvae and further in the head and trunk of 8-month-old larvae. These developmental stages correspond to the first detection of RAG1 and antigen-receptor (TCR and IgH) products in axolotl larvae. Our results suggest that in contrast to mammalian development, N diversity occurs early in axolotl development to diversify the primary repertoire. Phylogenetic analyses reveal that TdT and DNA polymerase (Pol ) genes are closely related, and that both enzymes were already present in the common ancestor of jawed vertebrates.This work is dedicated to the memory of Prof. Jacques CharlemagneThe sequences reported in this paper have been deposited in the GenBank database (accession numbers AF039209 and AY248700)     

Experimental hybridization between catfishes of the families Clariidae and Pangasiidae in Thailand

Synopsis Hybrids between the clariid speciesClarias macrocephalus andC. batrachus and the pangasiid speciesPangasius sutchi were obtained by hormone injection of brood stock and artificial fertilization. Pure parental crosses as well as all possible hybrid combinations were obtained. Fertility, hatchability, and post-yolk absorption survival was high (66–99%) in all pure parental crosses and in all crosses between the two species ofClarias. In crosses betweenClarias andPangasius fertilization was also very high (68–97%) but hatchability varied from 11 to 23% and post-yolk absorption survival from 0% inPangasius sutchi ×Clarias batrachus to about 50% inPangasius sutchi ×Clarias macrocephalus . The longest-lived hybrids ofPangasius andClarias were those ofPangasius sutchi ×Clarias macrocephalus which survived until the experiment was prematurely terminated due to contaminated food after 4 1/2 months, at which point they had grown to total lengths of 8–14cm. The hybrids comprised four morphotypes, two relativelyPangasius-like and two relativelyClarias-like, but all markedly different from the parental species.     

A short note onLecane species (rotifera) found inUtricularia minor vegetation

Summary InUtricularia minor vegetation a number ofLecane spp. are found. Remarks are made regarding confusion resulting from the use of VOIGT's keys. L.glypta is a synonym ofL.flexilis; L. lunaris andL.constricta belong to the species group L.lunaris andL.sinuata is a form ofL.hamata; L.hamata f.sinuata is new to the European fauna.     

Molecular evolution of mRNA: A method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its application

Summary A method for estimating the evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences is presented. This method is applied to genes of øX174 and G4 genomes, histone genes and-globin genes, for which homologous nucleotide sequences are available for comparison to be made. It is shown that the rates of synonymous substitutions are quite uniform among the non-overlapping genes of øX174 and G4 and among histone genes H4, H2B, H3 and H2A. A comparison between øX174 and G4 reveals that, in the overlapping segments of the A-gene, the rate of synonymous substitution is reduced more significantly than the rate of amino acid substitution relative to the corresponding rate in the nonoverlapping segment. It is also suggested that, in the coding regions surrounding the splicing points of intervening sequences of-globin genes, there exist rigid secondary structures. It is in only these regions that the-globin genes show the slowing down of evolutionary rates of both synonymous and amino acid substitutions in the primate line.     

Restriction fragment length polymorphism and evolution of the mouse immunoglobulin constant region gamma loci

Genomic DNA from twelve laboratory mouse strains, in addition to 21 wild-derived strains belonging to different taxa (Mus musculus domesticus, Mus musculus musculus, Mus spretus, Mus macedonicus, a and Mus spicilegus) and four mouse strains that are evolutionarily more distant, were analyzed by Southern blot for polymorphism of the Ig heavy chain constant region isotype (Igh-C) and for the distribution of the duplicated Igh-1 (C2) haplotype. Distinct allelic forms of each Igh-C locus could be defined by restriction fragment length polymorphism (RFLP). In laboratory mouse strains RFLP proved to be more sensitive in the detection of Igh-4 (C1) alleles than serological methods. Taq I digestion allowed the definition of two alleles in the Igh-8 (C3) locus, which is absolutely conserved at the protein levels. More extensive RFLP could be found in wild strains belonging to the subgenus Mus and in the evolutionarily more distant Mus species belonging to other subgenera. In previous studies we have shown that the Igh-1 locus is duplicated in M. m. musculus subspecies. We now extend this observations to the wild mouse strains belonging to M. spicilegus and M. macedonicus species and to the evolutionarily more distant wild mouse strain Mus pahari (subgenus coelomys), which is thought to have diverged from domestic mice about 5 million years ago. In addition, we found a similar RFLP pattern in ten of 18 wild mice trapped in India, suggesting that the haplotype containing the two Igh-1-like genes, organized in tandem as distinct isotypes, is widely spread in natural populations. The evolution of murine Igh-C-encoded isotypes is also discussed. Correspondence to: P.-A. Cazenave.     

Phylogeny and habitat adaptations within a monophyletic group of wetland moss genera (Amblystegiaceae)

Genus level phylogenetic patterns within a monophyletic group of wetland mosses consisting ofTomentypnum, Hamatocaulis, Scorpidium, Conardia, Calliergon, Warnstorfia, Straminergon, andLoeskypnum (Amblystegiaceae) are cladistically analysed, usingPalustriella and partlyCratoneuron as outgroups. The ingroup consists of two clades, one withTomentypnum, Hamatocaulis andScorpidium, the other with the other ingroup genera. The second clade gets completely resolved only with the inclusion of habitat data. The adaptation to relatively dry wetland habitats probably evolved in the ancestor ofStraminergon andLoeskypnum, the species ofCalliergon andWarnstorfia, which are more ancestral, growing in wetter habitats. The more primitive taxa of the ingroup, as well asPalustriella species, occur in relatively mineral-rich habitats and adaptations to poorer habitats occurred several times in the two clades.     

Nucleic acid hybridization of group N and group D streptococci

Streptococci of serological groups N and D were found to belong to three different 23S ribosomal RNA (rRNA) homology clusters which are not closely related to each other. One cluster includes the group N streptococci:Streptococcus lactis, S. cremoris, S. diacetylactis, and, in addition, two lactobacilli, Lactobacillus hordniae andL. xylosus. The second one is composed ofStreptococcus faecium, S. durans, andS. faecalis with its subspecies. All are members of serological group D. The third group includes the group D streptococciStreptococcus bovis andS. equinus together withS. thermophilus andS. salivarius. DNA-DNA hybridization studies confirm the close genetic relationship within each of the rRNA homology clusters.     

Synonymous nucleotide substitution rates of β-tubulin and histone genes conform to high overall genomic rates in rodents but not in sea urchins

Summary Sea urchin and rodent genomes have been posited to evolve rapidly as indicated by divergences in single copy nuclear DNA sequences. We have examined whether the synonymous substitution rates of three highly conserved genes, -tubulin, histone H4, and histone H3, adhere to these high genomic substitution rates by comparing sequences between two sea urchins,Strongylocentrotus purpuratus andLytechinus pictus, and between rodents and humans. Whereas the rate of change between the 3 untranslated regions of the -tubulin cDNA ofS. purpuratus (Sp-1), sequenced in this study, and ofL. pictus (Lp-3) was consistent with the overall rate of change estimated from previous DNA hybridization results between these species, the synonymous substitution rates for the carboxyl domains of these -tubulins, as well as for the late histones H4 and H3, were significantly depressed. In contrast, synonymous nucleotide substitution rates between rodents and between rodent and human for the carboxyl domain proper of identical -tubulin isotypes and for histone H4 and H3.1 did not differ from the overall rate of change for the rodent genomes. Moreover, an analysis of paralogous human and mouse -tubulin sequences supported the conclusion that the synonymous substitution rates in the mouse were higher than those in the human. Differences in constraint on evolutionary change were not evident strictly from the conserved amino acid sequences and base compositions of these genes. Other constraining influences seemed more relevant to the departure of the synonymous substitution rates of the sea urchin -tubulin and histone coding regions from the average genomic rate.     

Different ζ globin gene deletions among Black Americans

Summary Four types of chromosomes with a deletion between the human embryonic and globin genes were identified among 2.8% of 321 Black Americans from Georgia. Two deletions of approximately 11 kb which differed by about 300 bp occurred on chromosomes with or without a polymorphic Xba I site 5 to the globin gene [(X⁺) or (X^-)]. The deletions are identifiable in Xba I digests of genomic DNA using an or a globin gene probe which yield fragments of 23 kb from (X⁺)–^* chromosomes or 27 kb from (X^–)–^* chromosomes. Digestion with other enzymes and probing with both and probes gave fragments typical of the two globin gene deletions previously identified in Polynesians. Among Black Americans, these globin gene deletions have been found in combination with globin gene deletions in trans but not in cis. Homozygotes have not been found. Hematologic data on carriers of the globin gene deletions in association with Hb AS, SS, and SC suggest that these deletions have no effect on the function of the adult globin genes.