Isolation of DNA from yeasts

Methods are described that allow DNA to be prepared from widely different yeasts (Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50-150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.     

Isolation and characterization of repetitive DNA sequences from Panax ginseng

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization and evolution of the genome of interest. We report the isolation and characterization of the major classes of repetitive sequences from the genome of Panax ginseng. The isolation of repetitive DNA from P. ginseng was achieved by the reannealing of chemically hydrolyzed (200 bp-1 kb fragments) and heat-denatured genomic DNA to low C(o)t value. The low C(o)t fraction was cloned, and fifty-five P. ginseng clones were identified that contained repetitive sequences. Sequence analysis revealed that the fraction includes repetitive telomeric sequences, species-specific satellite sequences, chloroplast DNA fragments and sequences that are homologous to retrotransposons. Two of the retrotransposon-like sequences are homologous to Ty1/ copia-type retroelements of Zea mays, and six cloned sequences are homologous to various regions of the del retrotransposon of Lilium henryi. The del retrotransposon-like sequences and several novel repetitive DNA sequences from P. ginseng were used to differentiate P. ginseng from P. quinquefolius, and should be useful for evolutionary studies of these disjunct species.     

Isolation, characterization, and analysis of Leymus-specific DNA sequences.

Genomic Southern hybridization using labeled total genomic DNA of Leymus mollis as probe showed intense hybridization signals on all restriction enzyme digested DNA from five species of Leymus Hochst., and four species of Psathyrostachys Nevski. Experiments using the same L. mollis probe, but with unlabeled blocking DNA from Psathyrostachys, showed no hybridization at all. These two genera evidently had the same genomic content. Southern hybridization without blocking allowed identification of DNA fragments abundant in Leymus and Psathyrostachys. Fragments potentially specific to Leymus were cloned. Five repetitive DNA clones from L. mollis and L. arenarius were characterized: pLmIs1, pLmIs44, pLmIs51, pLmIs53, and pLaIs56. These clones hybridized to both Leymus and Psathyrostachys on Southern blots - no clone hybridized to only one of these genera. Both Southern blot and fluorescence in situ hybridization (FISH) experiments showed that all the clones contained dispersed repetitive sequences. They painted all and whole chromosomes uniformly except at centromeres, telomeres, and nucleolar organiser regions. Three of these clones, i.e., pLmIs1, pLmIs44, and pLmIs53, were essentially specific to Leymus and Psathyrostachys - little or no hybridization was detected in other genera such as Triticum, Hordeum, Thinopyrum, or Elymus. Sequence analysis further revealed that the clones were part of retroelements. In particular, the clone pLmIs44 produced hybridization profiles suitable for analysis of genetic relatedness among species. The present study shows that Leymus and Psathyrostachys share the same basic genome, Ns, and therefore provides strong evidence for combining these two genera.     

Isolation and characterization of DNA sequences from Triticum aestivum which function as origins of replication in Saccharomyces cerevisiae

Recombinant YIp5 plasmids with the DNA from Triticum aestivum are capable of autonomous replication in Saccharomyces cerevisiae. The URA transformants are unstable without selection pressure, and transformation of yeast cells with these plasmids occurs at high frequency. The cloned sequences were characterized and analyzed to state their belonging to Triticum tribe.     

Nematode repetitive DNA with ARS and segregation function in Saccharomyces cerevisiae.

Several members of a repetitive DNA family in the nematode Caenorhabditis elegans have been shown to express ARS and centromeric function in Saccharomyces cerevisiae. The repetitive family, denoted CeRep3, consists of dispersed repeated elements about 1 kilobase in length, present 50 to 100 times in the nematode genome. Three elements were sequenced and found to contain DNA sequences homologous to yeast ARS and CEN consensus sequences. Nematode DNA segments containing these repeats were tested for ARS and CEN (or SEG) function after ligation to shuttle vectors and introduction into yeast cells. Such nematode segments conferred ARS function to the plasmid, as judged by an increased frequency of transformation compared with control plasmids without ARS function. Some, but not all, also conferred to the plasmid increased mitotic stability, increased frequency of 2+:2- segregation in meiosis, and decreased plasmid copy number. These effects are similar to those of yeast centromeric DNA. In view of these results, we suggest that the CeRep3 repetitive family may have replication and centromeric functions in C. elegans.     

Isolation and characterization of DNA sequences from flow-sorted human chromosome 22 libraries

Two flow-sorted chromosome 22 libraries were used to isolate DNA sequences specific for chromosome 22. 45-phage DNAs were probed against human genomic DNA. 12 of them showed unique or low-copy character. Using digested DNA from rodent-human hybrid cell lines, 3 of the 12 recombinants were assigned unique to chromosome 22 and regionally mapped. 1 clone mapped to 22pter-q11, 1 clone to 22q12-qter and 1 clone, for which in situ hybridization was performed, to 22q13.1. 2 low-copy probes, 1 of them displaying polymorphisms in MspI and TaqI digests of individual DNAs, must have similar sequences on 22 and additional chromosomes. Furthermore, a highly repetitive DNA representing a compound locus of some hundred kilobases on chromosome 22 was isolated. These 6 probes may provide useful tools for studying the structure and function of this small chromosome involved in a relatively high number of inherited and acquired diseases.     

Isolation and characterization of genomic mouse DNA clones containing sequences homologous to tRNAs and 5S rRNA.

We have cloned and characterized three fragments of Balb/c mouse DNA which hybridize to mouse cell tRNAs. Fractionation of the tRNAs which hybridize to these clones reveals that two of the clones, lambda Mt-4A and lambda Mt-6A hybridize to only one or two tRNAs, while one clone, lambda Mt-4B, hybridizes to at least seven tRNAs. Two of the tRNAs were identified as tRNAProCCG and tRNAGlyGGA, and others have been identified as tRNAs which are selectively encapsidated into virions of murine leukemia virus and avian reticuloendotheliosis virus. The DNA sequences of putative genes for tRNAProCCG and tRNAGlyGGA, plus flanking regions, were determined. A clone of Balb/c mouse DNA which selectively hybridized to 5S rRNA was also isolated and partially characterized.     

Isolation and characterization of genome-specific DNA sequences in Triticeae species

Two contrasting genome-specific DNA sequences were isolated from Aegilops speltoides (wild goat grass) and Hordeum chilense (wild barley), each representing more than 1 % of the genomes. These repetitive DNA fragments were identified as being genome-specific before cloning by genomic Southern hybridization (using total genomic DNA as a probe), and hence extensive screening of clones was not required. For each fragment, up to six recombinant plasmid clones were screened and about half were genome-specific. Clone pAesKB52 from Ae. speltoides was a 763 by EcoRI fragment, physically organized in simple tandem repeats and shown to localize to sub-telomerec chromosome regions of species with the Triticeae S-genome by in situ hybridization to chromosomes. The sequence data showed an internal duplication of some 280 bp, which presumably occurred before sequence amplification and dispersion, perhaps by unequal crossing-over or reciprocal translocation. In situ hybridization showed that the sequence distribution varied between closely related (S-genome) species. Clone pHcKB6 was a 339 by DraI fragment from H. chilense, also tandemly repeated but more variable; loss of the DraI site resulting in a ladder pattern in Southern blots which had little background smear. In situ hybridization showed that the tandem repeats were present as small clusters dispersed along all chromosome arms except at a few discrete regions including the centromeres and telomeres. The clone hybridized essentially specifically to the H-genome of H. chilense and hence was able to identify the origin of chromosomes in a H. chilense x Secale africanum hybrid by in situ hybridization. It has a high A + T content (66%), small internal duplications, and a 50 by degenerate inverted repeat. We speculate that it has dispersed by retrotransposition in association with other sequences carrying coding domains. The organization and evolution of such sequences are important in understanding long-range genome organization and the types of change that can occur on evolutionary and plant breeding timescales. Genome-specific sequences are also useful as markers for alien chromatin in plant breeding.     

Isolation and characterization of nickel-accumulating yeasts

We selected three yeast strains that efficiently remove heavy metal ions from aqueous solution. We first screened yeasts that grew in the presence of 2 mM NiCl₂ among our stock of wild yeasts, and then selected those that removed Ni most efficiently from aqueous solution. These strains also removed Cu and Zn from aqueous solution and were identified as Candida species. Ni uptake was efficient at pH between 4.0 and 7.0, but less efficient at pH below 3.0. The amount of Ni taken up by the yeast cells was proportional to the initial concentration of NiCl₂ below about 4 mM Ni. The cells retained the abilities to remove Ni after treatment with 10 mM EDTA or 1 M HCl for repeated usage, or after heat treatment. Received: 16 December 1996 / Received last revision: 15 April 1997 / Accepted: 20 May 1997     

Isolation and chromosomal localization of genomic DNA sequences coding for cytoplasmic superoxide dismutase from Drosophila melanogaster

A synthetic oligodeoxynucleotide 18-mer probe derived from the amino acid sequence of Drosophila melanogaster cytoplasmic superoxide dismutase (cSOD) was used to screen a D. melanogaster genomic library. One of the positive clones maps by in situ hybridization to position 68A8-9 on the left arm of polytene chromosome 3, the region to which cSOD mutants have previously been mapped genetically. Partial sequence analysis verifies the presence of cSOD-coding sequences in this clone and indicates that the intron structure of the Drosophila cSOD gene differs significantly from its human counterpart.     

The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae.

Replication origins have been mapped to positions that coincide, within experimental error (several hundred base pairs), with ARS elements. To determine whether the DNA sequences required for ARS function on plasmids are required for chromosomal origin function, the chromosomal copy of ARS306 was deleted and the chromosomal copy of ARS307 was replaced with mutant derivatives of ARS307 containing single point mutations in domain A within the ARS core consensus sequence. The chromosomal origin function of these derivatives was assayed by two-dimensional agarose gel electrophoresis. Deletion of ARS306 deleted the associated replication origin. The effects on chromosomal origin function of mutations in domain A paralleled their effects on ARS function, as measured by plasmid stability. These results demonstrate that chromosomal origin function is a property of the ARS element itself.     

Isolation, characterization, and chromosomal location of the mouse enamelysin gene

Mouse enamelysin (Mmp20), a member of the matrix metalloproteinase (MMP) family of extracellular matrix degrading enzymes, shows a high degree of homology with other MMPs, particularly those of the stromelysin/collagenase subfamilies. It is expressed exclusively in ameloblasts and odontoblasts. The mouse enamelysin gene (Mmp20) is made up of 10 exons spanning approximately 65 kb within the MMP gene cluster at the centromeric end of chromosome 9.     

Isolation, characterization and chromosomal mapping of the mouse tyrosine aminotransferase gene

The tyrosine aminotransferase (TAT) gene is expressed in a tissue and developmental-specific manner. In addition, this gene is regulated by glucocorticoid and polypeptide hormones and its expression is affected when a regulatory region near the albino locus of the mouse is deleted. In order to allow studies of the molecular effects of these deletion mutations we have isolated and characterized the mouse TAT gene. The gene is 9.2 x 10(3) bases in length and consists of 12 exons which give rise to a 2.3 x 10(3) base long messenger RNA. The DNA sequence at the 5' end of the gene was determined and compared with the corresponding sequence of the rat tyrosine aminotransferase gene. The sequence comparison showed extensive homology over the entire region sequenced. In addition, DNA: DNA heteroduplex studies between the mouse and rat tyrosine aminotransferase genes revealed that this homology extends over the entire gene and its flanking sequences. The mouse tyrosine aminotransferase gene has been mapped distal to the serum esterase-1 locus on mouse chromosome 8, using a restriction fragment length polymorphism between two mouse species. Since the albino deletions are located on mouse chromosome 7, the assignment of the TAT gene to chromosome 8 suggests that a regulatory factor(s) affecting TAT gene expression acts in trans.     

Isolation and characterization of DNA from archaeological bone.

DNA was extracted from human and animal bones recovered from archaeological sites and mitochondrial DNA sequences were amplified from the extracts using the polymerase chain reaction. Evidence is presented that the amplified sequences are authentic and do not represent contamination by extraneous DNA. The results show that significant amounts of genetic information can survive for long periods in bone, and have important implications for evolutionary genetics, anthropology and forensic science.     

Molecular cloning and characterization of murine leukemia virus-related DNA sequences from C3H/HeN mouse DNA.

Ten murine leukemia virus (MuLV)-related DNA sequences were isolated from C3H/HeN mouse genomic DNA by cloning of EcoRI fragments in a Charon 4A vector. Detailed restriction endonuclease maps of four of the clones were developed by using AKR MuLV [32P]cDNA as a probe. C3H clone 14-9 contains approximately 7 kilobase pairs of MuLV-related DNA, one copy of an MuLV long terminal repeat-like sequence, and a region of flanking mouse DNA. C3H clones 34.2 and 36.1 contain approximately 2 kilobase pairs of MuLV-related DNA, one copy of a MuLV LTR-like sequence, and differing lengths of flanking mouse DNA sequences. C3H clone 8.13 was found to contain an insert of 5.7 kilobase pairs of MuLV-related DNA with two long terminal repeat-like regions and sequences which are partially homologous to AKv-1. Comparison fo the restriction endonuclease cleavage maps of these C3H clones with maps recently developed for ecotropic and xenotropic MuLV DNAs indicates that C3H clone 14-9 corresponds to the 5'-terminal portion of a genomic DNA sequence related to xenotropic MuLVs, whereas C3H clones 34.2 and 36.1 correspond to the 3' terminal portions of genomic DNA sequences related to xenotropic MuLVs. Clone 8.13 represents a deleted, xenotropic MuLV-related provirus. C3H clones 14-9, 34.2, 36.1, and 8.13 provide defined DNA sequence probes with which to characterize the organization and expression of endogenous MuLV-related DNA sequences in the mouse genome.     

Isolation of repeated and self-complementary sequences from E. coli DNA

We have used the single-strand specific nuclease from Neurospora crassa and chromatography on methylated albumin-kieselguhr to purify and characterize repeated and self-complementary sequences from Escherichia coli DNA. Approximately 0.5% of the genome renatures spontaneously at zero time and another 2% renatures somewhat more rapidly than the total DNA. The early renaturing DNA has a base composition and a Tm similar to the total DNA and contains on the average 100 base pairs; the self-complementary DNA also has a base composition like E. coli but contains a mean of 170 base pairs. No evidence was obtained for the presence of a highly redundant sequence.     

Isolation and characterization of ethanol-producing yeasts from fruits and tree barks

Aims:  Isolation and identification of yeasts converting xylose to ethanol.
Methods and Results:  A total of 374 yeasts were isolated from a variety of rotten fruits and barks of trees. Out of these, 27 yeast strains were able to assimilate xylose and produce 0·12–0·38 g of ethanol per gram of xylose. Based on phylogenetic analysis of D1/D2 domain sequence of LSU (Large Subunit) rRNA gene and phenotypic characteristics the ethanol-producing strains were identified as member(s) of the genera Pichia, Candida , Kluyveromyces, Issatchenkia, Zygosacchraomyces , Clavispora, Debaryomyces , Metschnikowia , Rhodotorula and Cryptococcus.
Conclusion:  Yeast strains producing ethanol from xylose have been isolated from a variety of rotten fruits and barks of trees and identified.
Significance and Impact of the Study:  Environmental isolates of yeasts which could convert xylose to ethanol could form the basis for bio-fuel production and proper utilization of xylan rich agricultural and forest wastes.     

Isolation and characterization of salmonid telomeric and centromeric satellite DNA sequences

Satellite DNA clones with a 37 bp repeat unit were obtained from BglII-digested genomic DNA of Masu salmon (Oncorhynchus masou) and Chum salmon (O. keta). Fluorescence in situ hybridization (FISH) analysis with the isolated clones as a probe showed that these repetitive sequences were localized in the telomeric regions of chromosomes in both species. Southern and dot blot analyses suggested conservation of homologous sequences with similar repeat unit in other salmonids including the species of the genus Oncorhynchus and Salvelinus, but lack or scarcity of such sequences in the genus Hucho and Salmo. Similarly, polymerase chain reaction (PCR)-based cloning of satellite DNA referring to a reported Rainbow trout (O. mykiss) centromeric sequence was successful for the Oncorhynchus, Salvelinus and Hucho species. The obtained satellite DNA clones were localized with FISH in the centromeric regions of chromosomes of the species from these three genera. Although PCR cloning of the centromeric satellite DNA had failed in the Salmo species due to some base changes in the priming sites, dot blot hybridization analysis suggested conservation of homologous satellite DNA in the genus Salmo as in the other three genera. In the neighbor-joining tree of cloned centromeric satellite DNA sequences, the genus Oncorhynchus and Salvelinus formed adjacent clades, and the clade of the genus Hucho included the reported centromeric sequence of the genus Salmo. Conservation pattern and molecular phylogeny of the telomeric and centromeric satellite DNA sequences isolated herein support a close phylogenetic relationship between the genus Oncorhynchus and Salvelinus and between the Salmo and Hucho.