Heterogeneity in the ribosomal RNA genes of the yeast Yarrowia lipolytica; cloning and analysis of two size classes of repeats

Southern blotting of DNA from the ascomycetous yeast Yarrowia lipolytica revealed two major size classes of DNA units coding for rRNAs, which differ in length by about 1000 bp. We have cloned an rDNA unit of each size class. R-looping experiments revealed that the rRNA genes of both units are uninterrupted; subsequent heteroduplex analysis showed that the size difference both units is located within the nontranscribed spacer. Sequence analysis revealed that a major part of these spacers consists of a complex pattern of repetitions in periodicities of up to about 150 bp and that the difference between both rDNA units are located mainly in this repetitive region. Apart from different lengths of the repetitive regions, both rDNA units also reveal extended microheterogeneity within their homologous parts. Furthermore, no gene for 5S rRNA was observed in the spacer region. Therefore, the organization of the spacer of Yarrowia rDNA is clearly different from that of Saccharomyces cerevisiae.     

High cell density culture of Yarrowia lipolytica using a one-step feeding process

Yarrowia lipolytica is a potentially useful host for heterologous protein production. To develop an efficient culture method for high cell density cultivation and heterologous gene expression of Y. lipolytica, the effects of medium components and their concentrations on the growth of Y. lipolytica have been investigated. Addition of yeast extract to the culture media was found to significantly reduce the long lag phase encountered when Y. lipolytica was cultivated in synthetic culture media containing high concentrations of glycerol. Therefore, by enriching with 0.3% yeast extract the synthetic culture medium containing 15% glycerol, we could cultivate Y. lipolytica up to 83 g/L dry cell weight in a batch culture. Furthermore, over 100 g/L and 88 units/mL of rice alpha-amylase activity were obtained in less than 50 h with a one-step feeding process in which a recombinant Y. lipolytica expressing rice alpha-amylase was cultivated in the 10% glycerol medium enriched with 0.3% yeast extract and fed only once with the concentrated feeding medium (60% glycerol). The easy cultivation of recombinant Y. lipolytica to a high cell density may strengthen its position as a host for heterologous protein production.     

Genomic arrangement of repeated PS700 elements in the nematode Panagrellus silusiae

When genomic DNA from the free-living nematode Panagrellus silusiae is digested with the restriction endonuclease BamHI and separated by electrophoresis, a band in the 700 base pair size range is evident after ethidium bromide staining. One of the 0.7-kilobase fragments (PS700-1) was characterized and found to be a member of a moderately repetitive DNA family (T. Warren and J.J. Pasternak. 1988. Nucleic Acids Res. 16: 10,833-10,847). In the current study, DNA sequence analyses of three independently isolated copies of the PS700 DNA family showed the same nucleotide sequence and greater than 98% similarity to PS700-1. Four EMBL-4 bacteriophage clones were isolated from a Panagrellus genomic DNA library with PS700-1 as the probe and were analyzed by restriction endonuclease site mapping and Southern blot DNA hybridization. These clones contain 31 copies of the PS700 DNA family. In each case, the units are arranged in head-to-tail arrays. One of the EMBL-4 clones contains copies of a novel variant of the PS700 elements. The maintenance of both nucleotide sequence and restriction endonuclease restriction site homogeneity among members of the dispersed PS700 DNA family may denote a functional role for these sequences.     

Structure and organization of the highly repeated and interspersed 1.3 kb EcoRI-Bg1II sequence family in mice.

EcoRI digestion of total mouse DNA yields a prominant 1.3 kb fragment amounting to between 1 and 2% of the mouse genome. The majority of the 1.3 kb EcoRI fragments have a single Bg1II site 800 bp from one end. This EcoRI-Bg1II sequence family shows HindIII and HaeIII sequence heterogeneity. We have cloned representatives of the EcoRI-Bg1II gene family in Charon 16A and studied their structure and organization within the genome. The cloned 1.3 kb fragments show the expected restriction enzyme patterns as well as additional heterogeneity. Representatives of the EcoRI-Bg1II sequence family were found to be interspersed throughout the mouse genome as judged by CsCl density gradient centrifugation experiments. Family members were also found to be organized in higher order repeating units. Homologous sequences were also found in other rodent species including rat and Chinese hamster. Cross hybridization between a cloned 1.3 kb mouse fragment and a cloned CHO repeated sequence is of special interest since the latter has been shown to contain sequences homologous to the Human A1uI family by nucleotide sequencing.     

Yarrowia lipolytica possesses two plasma membrane alkali metal cation/H+ antiporters with different functions in cell physiology

The family of Nha antiporters mediating the efflux of alkali metal cations in exchange for protons across the plasma membrane is conserved in all yeast species. Yarrowia lipolytica is a dimorphic yeast, phylogenetically very distant from the model yeast Saccharomyces cerevisiae. A search in its sequenced genome revealed two genes (designated as YlNHA1 and YlNHA2) with homology to the S. cerevisiae NHA1 gene, which encodes a plasma membrane alkali metal cation/H+ antiporter. Upon heterologous expression of both YlNHA genes in S. cerevisiae, we showed that Y. lipolytica antiporters differ not only in length and sequence, but also in their affinity for individual substrates. While the YlNha1 protein mainly increased cell tolerance to potassium, YlNha2p displayed a remarkable transport capacity for sodium. Thus, Y. lipolytica is the first example of a yeast species with two plasma membrane alkali metal cation/H+ antiporters differing in their putative functions in cell physiology; cell detoxification vs. the maintenance of stable intracellular pH, potassium content and cell volume.     

Subunit composition of mitochondrial complex I from the yeast Yarrowia lipolytica

Here we present a first assessment of the subunit inventory of mitochondrial complex I from the obligate aerobic yeast Yarrowia lipolytica. A total of 37 subunits were identified. In addition to the seven central, nuclear coded, and the seven mitochondrially coded subunits, 23 accessory subunits were found based on 2D electrophoretic and mass spectroscopic analysis in combination with sequence information from the Y. lipolytica genome. Nineteen of the 23 accessory subunits are clearly conserved between Y. lipolytica and mammals. The remaining four accessory subunits include NUWM, which has no apparent homologue in any other organism and is predicted to contain a single transmembrane domain bounded by highly charged extramembraneous domains. This structural organization is shared among a group of 7 subunits in the Y. lipolytica and 14 subunits in the mammalian enzyme. Because only five of these subunits display significant evolutionary conservation, their as yet unknown function is proposed to be structure- rather than sequence-specific. The NUWM subunit could be assigned to a hydrophobic subcomplex obtained by fragmentation and sucrose gradient centrifugation. Its position within the membrane arm was determined by electron microscopic single particle analysis of Y. lipolytica complex I decorated with a NUWM-specific monoclonal antibody.     

Structure of the major block of alphoid satellite DNA on the human Y chromosome

Alphoid DNA is a family of tandemly repeated simple sequences found mainly at the centromeres of the chromosomes of many primates. This paper describes the structure of the alphoid DNA at the centromere of the human Y chromosome. We have used pulsedfield gradient gel electrophoresis, cosmid cloning and DNA sequencing to determine the organization of the alphoid DNA on each of the Y chromosomes present in two somatic cell hybrids. In each case there is a single major block of alphoid DNA. This is approximately 470,000 bases (475 kb) long on one chromosome and approximately 575 kb long on the other. Apart from the size difference, the structures of the two blocks and the surrounding sequences are very similar. However, one restriction enzyme, AvaII, detects two clusters of sites within one block but does not cleave the other. The alphoid DNA within each block is organized into tandemly repeating units, most of which are about 5.7 kb long. A few variant units present on one chromosome are about 6.0 kb long. These variants, like the AvaII site variants, are clustered. The 5.7 kb and 6.0 kb units themselves consist of tandemly repeating 170 base-pair subunits. The 6.0 kb unit has two more of these subunits than the 5.7 kb unit. Our results provide a basis for further structural analysis of the human Y chromosome centromeric region, and suggest that long-range structural polymorphisms of tandemly repeated sequence families may be frequent.     

Tay1 protein, a novel telomere binding factor from Yarrowia lipolytica

Inspection of the complete genome of the yeast Yarrowia lipolytica for the presence of genes encoding homologues of known telomere-binding proteins surprisingly revealed no counterparts of typical yeast Myb domain-containing telomeric factors including Rap1 or Taz1. Instead, we identified a gene, YALIOD10923g, encoding a protein containing two Myb domains, exhibiting a high degree of similarity to the Myb domain of human telomeric proteins TRF1 and TRF2 and homologous to an essential fission yeast protein Mug152 whose expression is elevated during meiosis. The protein, which we named Tay1p (telomere-associated in Yarrowia lipolytica 1), was purified for biochemical studies. Using a model Y. lipolytica telomere, we demonstrate that the protein preferentially binds to Y. lipolytica telomeric tracts. Tay1p binds along the telomeric tract as dimers and larger oligomers, and it is able to remodel the telomeric DNA into both looped structures and synaptic complexes of two model telomere DNAs. The ability of Tay1p to induce dimerization of telomeres in vitro goes in line with its oligomeric nature, where each oligomer can employ several Myb domains to form intermolecular telomere clusters. We also provide experimental evidence that Tay1p may be associated with Y. lipolytica telomeres in vivo. Together with its homologues from Schizosaccharomyces pombe and several basidiomycetous fungi (Sánchez-Alonso, P., and Guzman, P. (2008) Fungal Genet. Biol. 45, S54-S62), Tay1p constitutes a novel family of putative telomeric factors whose analysis may be instrumental in understanding the function and evolution of double-stranded DNA telomeric proteins.     

Determination of restriction enzyme activity when cutting DNA labeled with the TOTO dye family

Optical mapping, a single DNA molecule genome analysis platform that can determine methylation profiles, uses fluorescently labeled DNA molecules that are elongated on the surface and digested with a restriction enzyme to produce a barcode of that molecule. Understanding how the cyanine fluorochromes affect enzyme activity can lead to other fluorochromes used in the optical mapping system. The effects of restriction digestion on fluorochrome labeled DNA (Ethidium Bromide, DAPI, H33258, EthD-1, TOTO-1) have been analyzed previously. However, TOTO-1 is a part of a family of cyanine fluorochromes (YOYO-1, TOTO-1, BOBO-1, POPO-1, YOYO-3, TOTO-3, BOBO-3, and POPO-3) and the rest of the fluorochromes have not been examined in terms of their effects on restriction digestion. In order to determine if the other dyes in the TOTO-1 family inhibit restriction enzymes in the same way as TOTO-1, lambda DNA was stained with a dye from the TOTO family and digested. The restriction enzyme activity in regards to each dye, as well as each restriction enzyme, was compared to determine the extent of digestion. YOYO-1, TOTO-1, and POPO-1 fluorochromes inhibited ScaI-HF, PmlI, and EcoRI restriction enzymes. Additionally, the mobility of labeled DNA fragments in an agarose gel changed depending on which dye was intercalated.     

Molecular characterization of Yarrowia lipolytica and Candida zeylanoides isolated from poultry

Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains of Yarrowia lipolytica and seven strains of Candida zeylanoides. Digestion of amplicons with HinfI and HaeIII produced two fragments of 200 and 150 bp from Y. lipolytica and three fragments of 350, 150, and 100 bp from C. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA of Y. lipolytica into three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained from C. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.     

Sequence analysis of Vicia faba highly repeated DNA: the BamHI repeated sequence families

Cleavage of Vicia faba nuclear DNA with the restriction endonuclease BamHI yielded discrete size classes of 250, 850, 900, 990, 1 150, 1 500 and 1 750 bp of highly repetitive DNA. Each of these sequence families comprised about 3% of the total genomic DNA. Some sequence members from each sequence family were cloned in pBR322 and their primary structures determined. Computer analyses of nucleotide sequences suggested the existence of about 60 bp sequence periodicity within the repeating unit of the 990 bp sequence family, though the extent of homology among the surmised shorter subrepeat units was very low. With other BamHI sequence families, however, the data did not show any clear internal sequence periodicity. The repeat units of the 850 bp and 1 750 bp sequence families contained nucleotide sequences homologous to the 250 bp family sequence. No sequence relationship between or among other sequence families was observed. There was 13–25% sequence variation among 6 cloned members of the 250 bp family and probably also among those of other BamHI repeat families. DNA sequences homologous to these V. faba BamHI repeat families were detected in Pisum sativum DNA by Southern blot hybridization. Furthermore, very weak cross-hybridization was observed with plant DNAs from Phaseolus vulgaris, Triticum aestivum, Cucumis sativus and Trillium kamtschaticum.     

Molecular genetic characterization of the RD-114 gene family of endogenous feline retroviral sequences.

RD-114 is a replication-competent, xenotropic retrovirus which is homologous to a family of moderately repetitive DNA sequences present at ca. 20 copies in the normal cellular genome of domestic cats. To examine the extent and character of genomic divergence of the RD-114 gene family as well as to assess their positional association within the cat genome, we have prepared a series of molecular clones of endogenous RD-114 DNA segments from a genomic library of cat cellular DNA. Their restriction endonuclease maps were compared with each other as well as to that of the prototype-inducible RD-114 which was molecularly cloned from a chronically infected human cell line. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. However, the env regions of many of the sequences examined were substantially deleted. Several of the endogenous RD-114 genomes contained a novel envelope sequence which was unrelated to the env gene of the prototype RD-114 env gene but which, like RD-114 and endogenous feline leukemia virus provirus, was found only in species of the genus Felis, and not in other closely related Felidae genera. The endogenous RD-114 sequences each had a distinct cellular flank which indicates that these sequences are not tandem but dispersed nonspecifically throughout the genome. Southern analysis of cat cellular DNA confirmed the conclusions about conserved restriction sites in endogenous sequences and indicated that a single locus may be responsible for the production of the major inducible form of RD-114.     

A new family of tandem repetitive early histone genes in the sea urchin Lytechinus pictus: evidence for concerted evolution within tandem arrays.

We have isolated and characterized a third nonallelic tandemly arrayed histone cluster (LpE) from the sea urchin Lytechinus pictus. Although this tandem array is not intermingled with the other two early histone gene families also found in the L. pictus genome, the order and polarity of the five histone coding sequences in this family are the same as every other well characterized sea urchin early histone gene family. Heteroduplex analysis and restriction endonuclease mapping experiments indicate that the LpE family is more closely related to the B-C than the A-D family of early histone genes. Examination of several individual sperm DNA samples has revealed considerable polymorphism in each of the three tandem repeat families. Within an individual, however, each family is remarkably homogeneous. Thus, our results indicate that rapid fixation of variants acts to homogenize the members of a single tandem array at a considerably faster rate within a family than between families. However, at least some exchange of sequences between families is evident based on the conservation of many restriction endonuclease recognition sites and from analysis of a a cosmid clone in which the A-D and E tandem repeats are found adjacent to one another. These differences in the rate of fixation of variants within and between these families are likely to be responsible for the maintenance of diversity between the different families.     

Restriction fragment-length polymorphisms of class II gene sequences in mice expressing minor structural variants of I-Ak and I-Ap

Serologic and structural analyses of the I-A molecules expressed among a large collection of wild mouse-derived H-2 haplotypes has led to the definition of "families" of I-A alleles which encode antigenically similar molecules that are identical in more than 90% of their tryptic peptides. Two of these families, denoted the I-Ak and I-Ap families, consist of 10 I-A alleles which encode I-A molecules whose structures are closely related to either I-Ap or I-Ak. The evolutionary relationships of the I-A alleles in these families were assessed by a molecular analysis of their genomic structures. The A alpha and A beta alleles within these I-A families were compared by analysis of restriction fragment-length polymorphisms (RFLP) detected at high stringency by Southern blot hybridization with DNA probes specific for either A alpha or A beta. The polymorphic restriction enzyme sites detected in this survey were distributed over more than 7 kb of genomic DNA surrounding each gene. Because both A alpha and A beta are encoded by about 700 bp of exon DNA, the majority of the restriction enzyme sites assayed by this RFLP analysis reflect polymorphisms in noncoding regions. The DNA sequence homologies of these alleles were estimated from the RFLP results with seven restriction endonucleases by calculating the fraction homologous value as defined previously. The results indicate that evolutionarily dissimilar I-A alleles can encode I-A molecules with very similar structures. The five I-A alleles in the I-Ak family could be divided into two discrete groups, denoted K1 and K2, on the basis of their restriction fragment (RF) genotypes. The RF genotypes of alleles within each group shared more than 80% of the restriction fragments for both A and A beta. In contrast, the RF genotypes of alleles in group K1 differed extensively from those in group K2, indicating that alleles in these separate groups may not be evolutionarily closely related. These observations suggest that gene conversion or intragenic recombinational events may have been involved in the evolution of groups K1 and K2 in the I-Ak family. The RF genotypes of alleles in the I-Ap family demonstrated a close evolutionary relationship among all but two of the alleles. These two alleles encoded I-A molecules whose structures were the least related to I-Ap of any of the alleles in the I-Ap family.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evolution of the T1 retroposon family in the Anopheles gambiae complex

The T1 family of retrotransposable elements is interspersed and moderately repeated in five member species of the Anopheles gambiae sibling-species complex and has diverged little since the radiation of the complex. T1 includes two closely related but independent subfamilies, defined by the presence or absence of linked sets of restriction sites, in all but one species, although the relative abundance of the subfamilies differs within each. Sequence analysis of a 349-bp region from 21 clones isolated from A. gambiae confirmed the bipartite organization by revealing 19 coordinated nucleotide differences between the two subfamilies--T1 alpha and T1 beta. Sequence divergence is not only greater between than within subfamilies, but divergence within T1 beta is less than that within T1 alpha. Between-species comparisons of genomic consensus restriction maps revealed that T1 alpha is fixed for species-diagnostic differences in all species. With one exception, these subfamilies account for approximately 70% of detectable T1 copies in the genome. The results support retroposition as the dominant mechanism underlying the evolution of the T1 family.     

Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species

Nicotiana tabacum (tobacco) is an allotetraploid derived from ancestors of the modern diploids, N. sylvestris and N. tomentosiformis. We identified and characterized two distinct families of 5S ribosomal DNA (rDNA) in N. tabacum; one family had an average 431 bp unit length and the other a 646 bp unit length. In the diploid species, N. sylvestris and N. tomentosiformis, the 5S rDNA unit lengths are 431 bp and 644 bp respectively. The non-coding spacer sequence of the short unit in tobacco had high sequence homology to the spacer of N. sylvestris5S rDNA, while the longer spacer of tobacco had high homology with the 5S spacer of N. tomentosiformis. This suggests that the two 5S families in tobacco have their origin in the diploid ancestors. The longer spacer sequence had a GC rich sub-region (called the T-genome sub-region) that was absent in the short spacer. Pulsed field gel analysis and fluorescent in situ hybridization to tobacco metaphase chromosomes showed that the two families of 5S rDNA units are spatially separate at two chromosomal loci, on chromosomes S8 (short family) and T8 (long family). The repeat copy number at each chromosomal locus showed heterogeneity between different tobacco cultivars, with a tendency for a decrease in the copy number of one family to be compensated by an increase in the copy number of the second family. Sequence analysis reveals there is as much diversity in 5S family units within the diploid species as there is within the T and S-genome 5S family units respectively, suggesting 5S diversification within each family had occurred before tobacco speciation. There is no evidence of interlocus homogenization of the two 5S families in tobacco. This is therefore substantially different to 18-26S rDNA where interlocus gene conversion has substantially influenced most sequences of S and T genome origin; possible reasons are discussed.