Presence of a 16S rRNA pseudogene in the bi-molecular plastid genome of the primitive brown alga Pylaiella littoralis. Evolutionary implications

Summary The plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm. is composed of two different circular DNA molecules: the largest carries two rrn operons, and the smallest, only one copy of both 16S and 23S rDNAs. 16S rDNA copies located on both molecules have been cloned and their nucleotide sequences determined: they are 65% homologous to one another. The expression of these genes was assayed by hybridizing in vivo labelled P. littoralis rRNAs to both clones, and specific oligonucleotides to total RNA from P. littoralis. Results indicate that the 16S rDNA copy located on the small molecule is a pseudogene. Comparisons of the functional gene with other 16S rRNA genes shows that chloroplasts from green plants emerged earlier from the cyanobacterial lineage than Euglena gracilis and Pylaiella littoralis plastids.     

Survey of repetitive sequences in <Emphasis Type="Italic">Silene latifolia</Emphasis> with respect to their distribution on sex chromosomes

We carried out a global survey of all major types of transposable elements in Silene latifolia, a model species with sex chromosomes that are in the early stages of their evolution. A shotgun genomic library was screened with genomic DNA to isolate and characterize the most abundant elements. We found that the most common types of elements were the subtelomeric tandem repeat X-43.1 and Gypsy retrotransposons, followed by Copia retrotransposons and LINE non-LTR elements. SINE elements and DNA transposons were less abundant. We also amplified transposable elements with degenerate primers and used them to screen the library. The localization of elements by FISH revealed that most of the Copia elements were accumulated on the Y chromosome. Surprisingly, one type of Gypsy element, which was similar to Ogre elements known from legumes, was almost absent on the Y chromosome but otherwise uniformly distributed on all chromosomes. Other types of elements were ubiquitous on all chromosomes. Moreover, we isolated and characterized two new tandem repeats. One of them, STAR-C, was localized at the centromeres of all chromosomes except the Y chromosome, where it was present on the p-arm. Its variant, STAR-Y, carrying a small deletion, was specifically localized on the q-arm of the Y chromosome. The second tandem repeat, TR1, co-localized with the 45S rDNA cluster in the subtelomeres of five pairs of autosomes. FISH analysis of other Silene species revealed that some elements (e.g., Ogre-like elements) are confined to the section Elisanthe while others (e.g. Copia or Athila-like elements) are present also in more distant species. Similarly, the centromeric satellite STAR-C was conserved in the genus Silene whereas the subtelomeric satellite X-43.1 was specific for Elisanthe section. Altogether, our data provide an overview of the repetitive sequences in Silene latifolia and revealed that genomic distribution and evolutionary dynamics differ among various repetitive elements. The unique pattern of repeat distribution is found on the Y chromosome, where some elements are accumulated while other elements are conspicuously absent, which probably reflects different forces shaping the Y chromosome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation and characterization of a Neurospora glucose-repressible gene

Summary Using differential hybridization, the cDNA copy of a Neurospora gene coding for an abundant glucose-repressible mRNA (grg-l) has been isolated. The cDNA was used to clone the genomic copy, and both were sequenced. The cDNA is nearly full length and contains putative translational start and termination codons. Conceptual translation indicates that grg-1 mRNA could direct the synthesis of a 7,000 molecular weight polypeptide. The genomic clone, contained in an 1,888 by PvuII fragment, encompasses the entire cDNA as well as 838 by of 5 and 369 bp of 3 flanking sequence. Comparison of the cDNA and genomic clones revealed the presence of two short introns in potential protein-coding sequences. grg-1 message levels were found to increase within minutes following the onset of glucose deprivation and rise 50 fold during the first 90 min of derepression.     

Cloning and characterisation of the ribosomal RNA genes of the dimorphic yeast,Yarrowia lipolytica

Summary The ribosomal RNA genes of Yarrowia lipolytica have been identified, both in restriction digests of total genomic DNA and in a pBR322 gene bank, by hybridisation with cloned Saccharomyces cerevisiae rDNA. The Y. lipolytica rDNA repeat unit is 8.9 kb in size and contains the genes for the 25S and 18S, but not the 5S, rRNA species. The number of copies of these repeat units is approx. 50 per haploid genome. Several clones were found which did not conform to the standard restriction map due to differences outside the coding region. It appears that there is either heterogeneity of the spacer sequence within a strain or that the Y. lipolytica rDNA genes may be present as a number of separate clusters within this yeast's genome.     

Identification,characterization and sequence of Candida albicans repetitive DNAs Rel-1 and Rel-2

Two moderately repetitive DNA elements, Rel-1 and Rel-2, were identified in a screen for clones that hybridized to a Candida albicans minichromosome. Rel-1, a 223-bp sequence, is C. albicans-specific. The 2789-bp Rel-2 sequence hybridizes weakly to C. stellatoidia DNA but not to DNA from several other yeast species. Genomic Southern-blot analysis indicated that Rel-1 and Rel-2 are often closely associated in the genome, suggesting that they may be subsequences of a larger repetitive element. Small subrepeats are located in the nucleotide sequence of both clones. Hybridization demonstrated that Rel-2 contains both repetitive and unique DNA sequences. The repetitive DNA is present on most, and perhaps all, C. albicans chromosomes. The unique sequence maps to chromosome 7; however, in some strains, it is also present on additional chromosomes.     

DNA repair genes of Saccharomyces cerevisiae: complementing rad4 and rev2 mutations by plasmids which cannot be propagated in Escherichia coli

Summary The RAD4 gene of yeast required for the incision step of DNA excision repair and the REV2 (= RAD5) gene involved in mutagenic DNA repair could not be isolated from genomic libraries propagated in E. coli regardless of copy number of the shuttle vector in yeast. Transformants with plasmids conferring UV resistance to a rad4-4 or a rev2-1 mutant were only recovered if yeast was transformed directly without previous amplification of the gene bank in E. coli. DNA preparations from these yeast clones yielded no transformants in E. coli but retransformation of yeast was possible. This lead to the isolation of a defective derivative of the rad4 complementing plasmid. The modified plasmid was now capable of transforming E. coli but still interfered significantly with its growth.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

Additional locus of rDNA sequence specific to the X chromosome of the liverwort, Marchantia polymorpha

The molecular cytogenetic organization of 17S ribosomal RNA genes (17S rDNA), a part of the 45S rDNA repeat, was investigated on the chromosomes of the liverwort Marchantia polymorpha using fluorescence in-situ hybridization (FISH). The numbers of 17S rDNA loci visualized in female and male chromosomes were ten and nine, respectively. This heterogeneous localization was due to the presence of an additional 17S rDNA locus on the X chromosome and its absence on the Y chromosome. The signal on the X chromosome covered almost the entire region of its long arm. The other nine signals were observed on the same loci of respective autosomes in both sexes. Southern hybridization analysis revealed an additional band including 17S rDNA exclusively on EcoRI digested female genomic DNA supporting the existence of an additional 17S rDNA locus on the X chromosome.     

Repetitive sequences upstream of the pfg27/25 gene determine polymorphism in laboratory and natural lines of Plasmodium falciparum

The structure of the genomic region located upstream of the gametocyte-specific gene pfg27/25 of Plasmodium falciparum was analysed in laboratory lines and field isolates of the parasite. The gene is located in a subtelomeric region of chromosome 13 in parasite clones 3D7 and HB3. Analysis of laboratory lines and field isolates of P. falciparum indicated that polymorphism upstream of pfg27/25 is mainly due to the structure of a repetitive DNA region located at about half a kilobase from the pfg27/25 coding sequence. Different types of repetitive sequences are present in this region, whose copy number is variable in different parasite lines. In addition a GC-rich sequence element contained in this region, which is proposed to be the startpoint of pfg27/25 mRNA, presents either a direct or a reverse orientation in different parasite lines. Genomic deletions upstream of the pfg27/25 gene are also described in two laboratory lines of the parasite, which eliminate two newly identified malaria genes, orf P and orf Gap, from the genome of these parasites. One of them, orf Gap, deleted from the reference parasite clone 3D7, is abundantly expressed as mature mRNA in asexual parasites. PCR analysis on 64 field isolates of P. falciparum indicated that orf P and orf Gap sequences are present in all tested samples of naturally propagating parasites.     

Length heterogeneity in ITS 2 and the methylation status of CCGG and GCGC sites in the rRNA genes of the genus Peronosclerospora

Summary The polymerase chain reaction (PCR) was used with primers complementary to conserved flanking sequences to amplify the internal transcribed spacer 2 (ITS 2) of the rDNA repeat units of five Peronoscleropora isolates, one each of P. sorghi, P. maydis, P. sacchari and tow of P. zeae. In contrast to the situation found in mostfungi that have been examined, length heterogeneity was evident in each sample. The rDNA composition of the amplified bands was confirmed by Southern hybridizations using an ITS 2 amplified from P. sorghi and cloned rDNA from Neurospora crassa as probes. Length heterogeneity was also detected in genomic DNA digests using the same probes. In addition to one dominant fragment for each isolate, there were several less frequent fragments of different sizes, and the isolate(s) for each species had a unique banding pattern for ITS 2. The absence of 5-methylcytosine residues in CCGG and GCGC sequences in the ribosomal genes of these four Peronosclerospora species was demonstrated by the production of identical banding patterns with ribosomal DNA probes following digestion of genomic DNA with MspI and HpaII, and by complete digestion with CfoI.     

Characterization of a new family of tobacco highly repetitive DNA,GRS, specific for theNicotiana tomentosiformis genomic component

Members of a new family of highly repetitive DNA sequences called GRS were isolated fromNicotiana tabacum L. genomic DNA and characterized. Cloned, sequenced monomeric units (180–182 bp) of GRS exhibit properties characteristic of molecules that possess a stable curvature. The GRS family represents about 0.15% of total genomic DNA (10⁴ copies per haploid genome) and could be derived from eitherNicotiana tomentosiformis orNicotiana otophora, two possible ancestors of the T genome of the amphidiploidN. tabacum. Sequence homology between the HRS60 (Koukalováet al. 1989) and the GRS family has been estimated to be 57%.In situ hybridization was used to localize GRS on mitotic chromosomes. Hybridization signals were obtained on five pairs of chromosomes at intercalary sites of the longer chromosome arms. The majority of GRS sequences appeared to be organized in tandem arrays and a minority were found to be dispersed through the genome in short clusters, interspersed with other types of DNA repeats, including 25S rDNA sequences. Several loci containing both GRS and HRS60 were also found. Such hybrid loci may indicate intergenomic transfer of the DNA in the amphidiploidN. tabacum. GRS sequences, like HRS60 (Fajkuset al. 1992), were found to specify the location of nucleosomes. The position of the nucleosome core has been mapped with respect to a conservedMbol site in the GRS sequence and an oligo A/T tract is a major centre of the DNA curvature.Dr Ann KentonWe were deeply saddened to hear of the death of our coauthor and friend, Dr Ann Kenton, on 1 September 1994. She worked in the Cytogenetics Section of the Jodrell Laboratory, Kew, since 1974. Her work for many years on the cytogenetics ofGibasis and other species in the Commelinaceae gave unique insight into the genus and provided a valuable framework for understanding chromosome evolution and divergence in many other genera. Recently, her studies of molecular cytogenetics in tobacco have enabled interpretation of the structure of the complex genomes in the genus.     

Organization of ribosomal RNA genes in Alternaria alternate Japanese pear pathotype,a host-selective AK-toxin-producing fungus

Summary DNA encoding ribosomal RNA (rRNA) of Alternaria alternata Japanese pear pathotype has been cloned in , replacement vector, , Fix. Restriction endonuclease mapping and Southern hybridization with the 18S and 28S rRNAs of Saccharomyces cerevisiae revealed the A. alternata rDNA to be tandemly repeating 8.15-kilobase pair unit. The restriction fragments of the unit were then subcloned in the plasmid vector Bluescribe M13- and partially sequenced. The determined sequences were compared with previously reported sequences of S. cerevisiae rRNAs and their genes. The locations of DNA sequences encoding the 5.8S, 18S, and 28S rRNAs were determined by homology search using reported sequences. The complete DNA sequence for 5.8S rRNA of the fungus was found to be highly conserved at more than 90 % homology in the fungi analyzed. However, sequence diversities were observed in limited regions involved in a helix structure, the helix (e), found at position 116–137.Deceased     

Molecular diversification of tandemly organized DNA sequences and heterochromatic chromosome regions in some triticeae species

The subtelomeric heterochromatin of rye (Secale cereale) chromosomes makes up 12–18% of the genome and consists largely of a small number of tandemly organized DNA sequence families. The genomic organization, chromosomal locations and the structural organization of monomer units of the major DNA sequences from these regions were investigated and compared in other Triticeae species from the generaSecale, Agropyron, Dasypyrum, Triticum andHordeum. Southern hybridization and polymerase chain reaction analysis established that all studied species preserve the tandem type of sequence organization but the copy number is altered drastically between species. In the pSc200 family, a fraction of the tandem arrays is present with a head-to-head orientation of dimers inS. cereale andS. montanum. Members of the same family are more heterogeneous and present as head-to-head monomers in theDasypyrum species andA. cristatum. In situ hybridization demonstrates different organization of the sequence families in the various species: pSc200 and pSc250 are concentrated in major blocks at the ends of most rye chromosome arms, whereas they are more dispersed and in smaller blocks inDasypyrum andAgropyron indicating that accumulation is not simply due to the sequence itself. In contrast to rye,D. villosum has large blocks of only pSc200 whereasD. breviaristatum shows greater amplification of pSc250. These data indicate that each repetitive family is an independent unit of evolution, and suggest that the twoDasypyrum species are not closely related. The data are discussed in terms of existing evolutionary models for repetitive DNA sequences. The contribution of random events, through molecular drive and selection, to the evolution of heterochromatic regions is considered.accepted for publication by S. Mizuno     

Multiple-copy integration in the yeast Yarrowia lipolytica

Using an EcoRI-BglII fragment of the G unit of the rDNA of Y. lipolytica and a set of 11 deletions in the URA3 promoter, we have constructed several plasmids to test gene amplification in the rDNA. These plasmids contain the rDNA fragment for integration, defective versions of the URA3 gene, the XPR2 gene encoding alkaline extracellular protease (AEP) as a reporter gene, and part of the pBR322 plasmid for selection and replication in E. coli. Among these plasmids, one corresponds to a deletion which allows multiple integration into the rDNA (plasmid pINA773). Two other plasmids (pINA767 and pINA772) give multiple integration only with a mutated URA3 gene. Transformants carrying these three plasmids were tested for copy number, stability, chromosomal localization and AEP secretion. Transformants containing plasmids pINA767, 772 and 773 displayed an average copy number of 5, 12 and 25–60 copies respectively of the plasmid, as estimated by PCR and DNA hybridization. Integrations occurred in only one chromosome except for transformants containing 60 copies where copies were observed at least in two different chromosomes. Multiple integrations were found both as tandem repeats and as dispersed copies. Plasmid copy number was stable in both minimum and rich media, for strains containing less than ten copies per cells. However, for higher copy number, multiple integrations were stable only when AEP synthesis was not induced, while in inducing medium stability of the multiple integrations was dramatically affected.     

Rapidly evolving repetitive DNAs in a conservative genome: A test of factors that affect chromosomal evolution

The hypothesis that tandemly repeated DNA sequences may facilitate chromosomal rearrangements was tested by comparing a conservatively evolving karyotype of a bat species (Macrotus waterhousii) with data published for a rapidly evolving karyotype of an equid species (Equus zebra). Empirical data generated from the phylogenetic screening of rapidly evolving repetitive DNAs from approximately 0.1% of theM. waterhousii genome showed only one sequence that was repetitive inM. waterhousii but low in copy number or absent from the outgroupArtibeus jamaicensis. This compares to 34 such clones containing sequences which were repetitive inE. zebra but were low in copy number or absent from the outgroupCeratotherium simum. The bat sequence represents a single family of repeated sequences, whereas six families of sequences were identified inE. zebra. Southern blot analysis suggested that the sequence fromM. waterhousii is interspersed rather than tandemly repeated, as are the sequences inE. zebra. These data support the above hypothesis and suggest that species with conservatively evolving karyotypes have fewer numbers and families of rapidly evolving DNA sequences than do species such as the equids that possess a karyotype that is considered to have undergone rapid karyotypic evolution.     

Molecular and cytogenetic characterization of site-specific repetitive DNA sequences in the Chinese soft-shelled turtle (<Emphasis Type="Italic">Pelodiscus sinensis</Emphasis>, Trionychidae)

A novel family of repetitive DNA sequences that are components of constitutive heterochromatin were cloned from BglI-digested genomic DNA of the Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae), and characterized by filter hybridization and chromosome in-situ hybridization. The BglI-family of repetitive sequences were classified into four types by their genome organization and chromosomal distribution as follows: the repeated sequences located on (1) two pairs of microchromosomes, (2) four pairs of microchromosomes,(3) about half the number of microchromosomes and (4) the interstitial region of the short arm of chromosome 2. The presence of microchromosome-specific repetitive sequences has also been reported in the Struthioniformes and Galliformes, suggesting that turtle chromosomes retain some similarity to the chromosome structure as well as the karyotypes of avian species     

Organization of ribosomal RNA genes in the fungus Cochliobolus heterostrophus

Summary The genes encoding the 17S, 5.8S and 25S ribosomal RNAs in the Ascomycete Cochliobolus heterostrophus were cloned and analyzed. They are arranged in tandemly repeated units (rDNA) either 9.0 or 9.15 kilobases in length, depending upon the strain. The 5S rRNA genes are not part of the tandemly repeated rDNA. Instead, many and perhaps all of the 5S genes are dispersed in the genome, as they are in the fungi Neurospora, Aspergillus and Schizosaccharomyces. Comparative restriction maps of the rDNA from C. heterostrophus and other filamentous fungi and yeasts are presented. A survey of rDNAs from twenty-three field isolates of C. heterostrophus collected worldwide demonstrated that each isolate has one or the other of two rDNA forms, which differ in length and in the presence or absence of at least three restriction enzyme sites. The differences are all located in spacer DNA outside the coding regions for the rRNA genes. Heterogeneity in the rDNA repeat was not observed within any single isolate. The copy number of the rRNA gene cluster in C. heterostrophus is approximately 130 per haploid genome.     

Structure and Organization of the rDNA Intergenic Spacer in Lake Trout (Salvelinus namaycush)

A total-genomic cosmid library was created to isolate complete copies of the rDNA cistron of lake trout (Salvelinus namaycush) in order to study the structure and organization of the intergenic spacer (IGS) in this species. A total of 60 rDNA- positive clones (average inserts > 25 kb) was recovered by screening the library with a rDNA-specific probe. Positive clones were assayed for the presence of the two internal rDNA spacers (ITS-1 and ITS-2) and the entire IGS fragment was successfully amplified from 42 clones by PCR. Length of the IGS fragments ranged from 9.4 to 17.8 kb. Comparative restriction mapping of the IGS–PCR products of several clones indicated two regions of extensive length variation surrounding a central region with sequence conservation. DNA sequence analysis was used to investigate the molecular basis of the IGS length variation and focused on identifying the region responsible for this variation. Over 9 kb of DNA sequence was obtained for one clone (A1) with a total IGS length of approximately 12.4 kb. Sequence of a conserved central region contained two open reading frames and a number of short direct repeats. Length variation in the IGS was determined by RFLP to result from differences in the number of copies of repetitive DNA sequences. These included an 89-bp tandem repeat ( repeats), an 82-bp element ( repeats), a 168–177-bp element (S repeats), and a 179–201-bp element ( repeats). Overall nucleotide composition of the IGS was biased towards A and T (%GC = 47.4). Maintenance of discrete rDNA-length variants in lake trout suggests that the rate of gene conversion is insufficient to produce homogeneous copies across the genome.     

A novel family of repetitive DNA sequences amplified site-specifically on the W chromosomes in Neognathous birds

A novel family of repetitive DNA sequences was molecularly cloned from ApaI-digested genomic DNA of two Galliformes species, Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris), and characterized by chromosome in-situ hybridization and filter hybridization. Both the repeated sequence elements produced intensely painted signals on the W chromosomes, whereas they weakly hybridized to whole chromosomal regions as interspersed-type repetitive sequences. The repeated elements of the two species had high similarity of nucleotide sequences, and cross-hybridized to chromosomes of two other Galliformes species, chicken (Gallus gallus) and blue-breasted quail (Coturnix chinensis). The nucleotide sequences were conserved in three other orders of Neognathous birds, the Strigiformes, Gruiformes and Falconiformes, but not in Palaeognathous birds, the Struthioniformes and Tinamiformes, indicating that the repeated sequence elements were amplified on the W chromosomes in the lineage of Neognathous birds after the common ancestor diverged into the Palaeognathae and Neognathae. They are components of the W heterochromatin in Neognathous birds, and a good molecular cytogenetic marker for estimating the phylogenetic relationships and for clarifying the origin of the sex chromosome heterochromatin and the process of sex chromosome differentiation in birds.     

Nucleotide sequence of the Schizosaccharomyces japonicus var. versatilis ribosomal RNA gene cluster and its phylogenetic implications

Fission yeasts form a small but heterogeneous group of ascomycetes and it is still unclear whether they should be subdivided into three genera (Schizosaccharomyces, Octosporomyces, Hasegawaea) or remain a single genus (Schizosaccharomyces). In order to decide whether a new genus Hasegawaea should be established for the species Schizosaccharomyces japonicus and Schizosaccharomyces versatilis, we have characterized the entire rDNA cluster in Schizosaccharomyces japonicus var. versatilis and compared it with the homologous region from Schizosaccharomyces pombe and with complete rRNA gene sequences from other yeast genera. From a phage genomic library a recombinant lambda phage containing the entire rDNA repeat unit was isolated. In this paper we report the primary sequence of the 18s, 5.8s and 25s rRNA coding regions. The S. japonicus var. versatilis rRNA genes are 1823 (18s), 158 (5.8s) and 3422 (25s) nucleotides long. The two sequences of the larger rRNA genes exhibit 95.7% (18s) and 93% (25s) similarity with the homologous genes from S. pombe. The differences between the rRNA genes of S. japonicus and S. pombe, however, are much smaller than the intrageneric differences within the rDNA sequences of other yeast genera. Therefore, subdivision of fission yeasts into the genera Schizosaccharomyces and Hasegawaea does not to seem to be justified. The sequence has been deposited in the EMBL data bank under the accession number Z 32848.