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  • 1
    ISSN: 1432-1432
    Keywords: Key words: DNA sequence —Gonium pectorale— Internal transcribed spacer — Intron — Mating type — Phylogeny — Volvocales
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. The morphologically uniform species Gonium pectorale is a colonial green flagellate of worldwide distribution. The affinities of 25 isolates from 18 sites on five continents were assessed by both DNA sequence comparisons and sexual compatibility. Complete sequences were obtained (i) for the internal transcribed spacer ITS-1 and ITS-2 regions of ribosomal DNA and (ii) for each of three single-copy spliceosomal introns, two in a small G protein and one in the actin gene. ITS sequences appeared to homogenize sufficiently rapidly to behave as a single copy gene. Intron sequence differences between isolates in this species reached nucleotide substitution saturation, while ITS sequences did not. Parsimony and evolutionary distance analysis of the two types of DNA data gave essentially the same tree conformation. By all these criteria, the group of G. pectorale isolates fell into two main clades, A and B. Clade A, with isolates from four continents, was comprised of four subclades of quite closely related isolates, plus one strain of ambiguous affinity. Clade B was comprised of two subclades represented by South African and South American isolates, respectively; thus, only subclades of clade B showed geographical localization. With respect to mating, all isolates except one homothallic strain and one apparently sterile strain fell into either one or the other of two mating types. Pairings in all possible combinations revealed that isolates from the same site formed abundant zygotes, which germinated to produce new, sexually active organisms. Zygotes were also formed in many pairings of other combinations, including crosses of clade A with clade B organisms, but none of the latter produced viable germlings. The ability to mate and produce viable progeny that were themselves capable of sexual reproduction was restricted to members of subclades established on the basis of DNA sequence similarities. Thus, the grades of difference in both nuclear intron sequences and rDNA ITS sequences paralleled those observed in the sexual analysis.
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  • 2
    ISSN: 1432-0983
    Keywords: Green algae ; Volvox ; Transcription signals ; Gene expression ; Intron ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The genome of the green alga Chlamydomonas reinhardtii contains approximately 15 gene clusters of the nucleosomal (or core) histone H2A, H2B, H3 and H4 genes and at least one histone H1 gene. Seven non-allelic histone gene loci were isolated from a genomic library, physically mapped, and the nucleotide sequences of three isotypes of each core histone gene species and one linked H1 gene determined. The core histone genes are organized in clusters of H2A–H2B and H3–H4 pairs, in which each gene pair shows outwardly divergent transcription from a short (300 bp) intercistronic region. These intercistronic regions contain typically conserved promoter elements, namely a TATA-box and the three motifs TGGCCAG-G(G/C)-CGAG, CGTTGACC and CGGTTG. Different from the genes of higher plants, but like those of animals and the related alga Volvox the 3′ untranslated regions contain no poly A signal, but a palindromic sequence (3′ palindrome) essential for mRNA processing is present. One single H1 gene was found in close linkage to a H2A–H2B pair. The H1 upstream region contains the octameric promoter element GGTTGA-CC (also found upstream of the core histone genes) and two specific sequence motifs that are shared only with the Volvox H1 promoters. This suggests differential transcription of the H1 and the core histone genes. The H1 gene is interrupted by two introns. Unlike Volvox H3 genes, the three sequenced H3 isoforms are intronfree. Primer-directed PCR of genomic DNA demonstrated, however, that at least 8 of the about 15 H3 genes do contain one intron at a conserved position. In synchronized C. reinhardtii cells, H4 mRNA levels (representative of all core histone mRNAs) peak during cell division, suggesting strict replication-dependent gene control. The derived peptide sequences place C. reinhardtii core histones closer to plants than to animals, except that the H2A histones are more animal-like. The peptide sequence of histone H1 is closely related to the V. carteri VH1-II (66% identity). Organization of the core histone gene in pairs, and non-polyadenylation of mRNAs are features shared with animals, whereas peptide sequences and enhancer elements are shared with higher plants, assigning the volvocalean histone genes a position intermediate between animals and plants.
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  • 3
    ISSN: 1432-0983
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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  • 4
    ISSN: 1432-0983
    Keywords: ypt genes ; rab gene family ; Gene expression ; Nucleotide sequence ; GTP-binding ; Ras superfamily ; Phylogeny ; Green algae ; Volvocales
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In addition to the previously described gene yptV1 encoding a small G-protein we have now identified and sequenced four more ras-related ypt genes (yptV2-yptV5) from the green alga Volvox carteri. The four new genes encode polypeptides consisting of 203 to 217 amino-acid residues that contain the typical sequence elements (GTP-binding domains, effector domain) of the ypt/rab subgroup of the Ras superfamily. Comparison of the derived amino-acid sequences from the V. carteri ypt gene products and their Ypt homologs from other species revealed similarity values ranging from 60% to 85%, whereas intraspecies similarities were found to approach only 55%. The coding sequences are interrupted by 5–7 introns of variable size (70–1000 nucleotides) occupying different positions in the genes. Reverse-transcribed samples of stage-specific RNAs were PCR-amplified with primers specific to yptV1, yptV3, yptV4, and yptV5 to determine if yptV transcription might be restricted to either cell type or to a specific stage of the life cycle. These experiments demonstrated that each of these genes is expressed throughout the entire Volvox life cycle and in both the somatic and the reproductive cells of the alga. The transcription start sites of yptV1 and yptV5 were mapped by primer extension. Expression of recombinant yptV cDNA in E. coli yielded recombinant proteins that bound GTP specifically, demonstrating a property which is typical for small G-proteins. The derived YptV polypeptide sequences were used to group them into four distinct classes of Ras-like proteins. These are the first proteins of the Ras superfamily to be identified in a green alga. We discuss the possible role of the YptV-proteins in the intracellular vesicle transport of Volvox.
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Current genetics 26 (1994), S. 497-505 
    ISSN: 1432-0983
    Keywords: Intron evolution ; Introns-early ; Introns-late ; Exon shuffling ; Ras family ; Green algae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Because all small G proteins (SGPs) possess a very similar array of structural and functional domains, they are obvious candidates for examining the relationships postulated to exist between the exon-intron structure of genes and the domain structure of the encoded proteins. To address this issue, and to possibly gain insight into the evolution of their introns, we have analyzed positions, sizes, and sequences of 125 introns from 28 SGP genes. These introns were found to be distributed in 60 different locations throughout the aligned sequences, with a preference for the 5′-half of the genes. More than 50% of the positions were found to be shared by two or more genes, and genes encoding SGPs of very similar amino acid sequence (i.e., isotypes) in quite closely related species tend to have most, or all, of their introns in identical locations, indicating a common evolutionary origin (homologous introns). However, with few exceptions, no statistically significant sequence similarity or common folding motif was found between homologous intron pairs. Only three intron positions are shared between members of distantly related SGP subfamilies. These three potentially ancient intron locations fall between regions encoding α-helices or β-sheets, but two of them interrupt regions encoding known functional (guanosine-nucleotide-binding) modules. Intron positions that are occupied only in single genes, or in genes encoding very similar SGPs, do not show any preferential distribution with respect to regions encoding structural or functional motifs. This discordance between exon modules and structural and/or functional protein domains suggests that most, if not all, introns in modern SGP genes arose by independent insertion events after diversification of the various SGP subfamilies, and therefore probably did not participate in the early evolution of these genes.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 31 (1996), S. 279-293 
    ISSN: 1573-5028
    Keywords: Ras superfamily ; Ypt/Rab proteins ; Volvox carteri ; Chlamydomonas reinhardtii
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Ypt/Rab proteins are small GTPases, which belong to the Ras superfamily and have been shown to be involved in endo-and exocytosis in mammalian cells and yeast. Using affinity-purified antibodies specific for four Ypt proteins, namely Ypt1p, Ypt4p, Ypt5p and Ypt6p, of the multicellular green alga Volvox carteri (YptVp) and its close unicellular relative Chlamydomonas reinhardtii (YptCp), we examined the abundance of the corresponding antigens during the asexual life cycle of Volvox, and their intracellular localization. The YptV proteins were found in all stages throughout the asexual life cycle and are tightly associated with intracellular membranes. Indirect immunofluorescence revealed that YptV4p, YptV5p and YptV6p are present in perinuclear regions of the cell, indicating an association with the Golgi region. Golgi localization of YptV4p and YptV6p in Volvox was confirmed by immunogold electron microscopy. In contrast, we found Ypt1p associated with the contractile vacuole in both V. carteri and C. reinhardtii. Furthermore, the YptV proteins were also detected along the entire length of the flagella of somatic Volvox cells. This flagellar location was substantiated by western blot analysis of extracts prepared from isolated flagella of both algae. While localization to exocytic compartments is in agreement with the established Ypt/Rab function in intracellular vesicle transport of eukaryotic cells, presence in the algal flagellum is the first hint of a possible role for small G proteins also in motility organelles.
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  • 7
    ISSN: 1615-6102
    Keywords: ypt/rab genes ; GTPase-activating protein ; Differentiation ; Ras superfamily ; Chlamydomonas
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The multicellular fresh-water algaVolvox carteri contains at least six small G protein-encoding genes (ypt genes) whose products are probably involved in intracellular vesicle transport. Four of them, YptV1, YptV3, YptV4, and YptV5, have been isolated and characterized previously. Here we report the cloning ofyptV2 cDNA, the production of recombinant His-tagged YptV2p protein (reYptV2p) inE. coli, and the analysis of its GTPase activity and intracellular localization. YptV2p is predominantly present in dividingVolvox embryos. It is a membrane-associated protein which is localized to the cell periphery (plasma membrane or plasma-membrane-associated vesicles), probably by a lipid moiety. Purified,E. coli-expressed YptV2p binds GTP specifically, and has a typically low intrinsic GTPase activity (kcat=0.004/min), which is enhanced by a GTPase activating protein activity present inVolvox. Our observations suggest a role of YptV2p in secretion, with a peak during the rapid cleavages of theVolvox embryo.
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  • 8
    ISSN: 1617-4623
    Keywords: rab genes ; Ras superfamily ; Algae Yeast ; Protein structure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract cDNAs representing nine small G protein genes encoding Ypt proteins from the green algae Volvox carteri (YptV) and Chlamydomonas reinhardtii (YptC) were tested for their ability to complement mutations in the YPT1, SEC4, and YPT7 genes of Saccharomyces cerevisiae strains defective in different steps of intracellular vesicle transport. None of the heterologously expressed algal genes was able to complement mutations in SEC4 or YPT7, but three of them, yptV1, yptC1, and yptV2, restored a YPT1 null mutation. On the amino acid sequence level, and particularly with respect to known small G protein specificity domains, YptVlp and YptVlp are the closest algal analogs of yeast Yptlp, with 70% overall identity and identical effector regions, but YptV2p is only 55% identical to Yptlp, and its effector domain resembles that of Sec4p. To define more precisely the regions that supply Yptlp function, six chimeras were constructed by reciprocal exchange of 68/72-, 122/123-, and 162/163-amino acid segments of the C-terminal regions between YptVlp (complementing) and YptV3p (non-complementing). Segments containing 68 amino acids of the hypervariable C-terminal, and 41 residues of the N-terminal region including the effector region, of YptVlp could be replaced by the corresponding parts of YptV3p without loss of function in yeast, but exchanges within the central core destroyed the ability to rescue the YPT1 mutation. Sequence analysis of ypt1-complementing and -noncomplementing Ypt types suggests that surface loop3 represents a novel specificity domain of small G proteins.
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  • 9
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Biologie in unserer Zeit 26 (1996), S. 179-186 
    ISSN: 0045-205X
    Keywords: Life and Medical Sciences
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Selten gab es in der Geschichte der Molekularbiologie ein besseres Beispiel für die Fruchtbarkeit interdisziplinärer Zusammenarbeit als die Aufklärung der Transportprozesse, die zwischen intrazellulären Membrankompartimenten ablaufen. Endoplasmatisches Reticulum. Golgi-Apparat, Endosomen, Vakuolen und die Plasmamenbran, die das Cytoplasma eukaryotischer Zellen eingrenzen und in charakteristische Kompartimente mit spezifischen Aufgaben aufteilen, müssen ständig miteinander und mit der Umwelt interagieren. Nach einem weitgehend akzeptierten Modell wird diese Aufgabe im wesentlichen durch einen Transport erfüllt, der aus einem gerichteten Fluß kleiner, membranumhüllter Vesikel besteht. Diese Vesikel verbinden die Kompartimente, indem sie ihren Inhalt jeweils von einem Donorkompartiment aufnehmen, sich abschnüren, zum Zielort wandern und mit einem Akzeptorkompartiment verschmelzen. Knospung, Zielfindung, Andocken und Fusion von Membranvesikeln sind demnach die Schlüsselprozesse des intrazellulären Transports. Den zugrundeliegenden molekularen Mechanismen gilt derzeit viel Aufmerksamkeit. Grundlegende Arbeiten aus den Bereichen Zellbiologie, Biochemie und Molekulargenetik haben ergänzend zusammengewirkt, um die zentralen Komponenten der intrazellulären Membrantransportmaschinerie in vitro und in vivo zu charakterisieren. Das organisierte molekulare Zusammenspiel wird durch die „SNARE-Hypothese“ von James Rothman elegant erklärt. Doch es gibt auch widersprüchliche Befunde, die auf nichtvesikuläre Transporte hindeuten und somit zeigen, daß der membranvermittelte Proteintransport vermutlich eine viel höhere Komplexität besitzt.
    Additional Material: 5 Ill.
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  • 10
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Biologie in unserer Zeit 25 (1995), S. 44-50 
    ISSN: 0045-205X
    Keywords: Life and Medical Sciences
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Die kleinen G-Proteine gehören zu jenen Enzymen, deren Vielfalt nach dem Entdeckungsboom, der mit dem Proto-Onkogen Ras vor zehn Jahren begann, auch heute noch kaum annähernd beschrieben sein dürfte. Kleine G-Proteine gibt es in allen Eukaryoten, und obgleich sie einen einfachen Aufbau haben und ihre enzymatische Aktivität primär lediglich in Bindung und Spaltung von Guanosintriphosphat besteht, sind ihre darauf basierenden Funktionen ebenso vielfältig wie die zellulären Prozesse, in die sie eingeschaltet sind: Die verschiedenen Vertreter kleiner G-Proteine sind an Signalketten bei der Steuerung der Zelldifferenzierung, beim Aufbau des Zellskeletts, bei der Aufrechterhaltung der Zellpolarität, bei der Kontrolle von Zellteilung, DNA- und RNA-Stoffwechsel und am Transport von Membranvesikeln beteiligt. Ihre detaillierte Untersuchung ist in den letzten Jahren zu einer wesentlichen Herausforderung der molekularbiologischen Forschung geworden.
    Additional Material: 6 Ill.
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