Characterization of the genome of the basidiomycete Schizophyllum commune

DNA of Schizophyllum commune was isolated both from mycelial cells and from protoplasts. Nuclear DNA was isolated after solubilization of the mitochondria with the detergent Nonidet. The G + C content of the nuclear DNA was 57%, calculated from its buoyant density (1.7165 g/ml) and from the Tm (77.4 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). The buoyant density of the ribosomal cistrons was 1.707 g/ml. DNA isolated from purified mitochondria had a very low G + C content: 22% (rho = 1.6845 g/ml, Tm = 61.8 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). Analysis of CsCl profiles and melting patterns suggested that mitochondrial DNA contains interspersed (A + T)-rich sequences. From reassociation analysis of sheared nuclear DNA the genome size of S. commune was determined to be 22.8 . 10(9) daltons. A small amount of DNA (0.5 . 10(9) daltons) bound to hydroxyapatite at zero time Cot. 7% of the genome (1.6 . 10(9) daltons) represented repetitive DNA.     

Absence of short-period repetitive-sequence interspersion in the Basidiomycete Schizophyllum commune

Reassociation kinetics and electron microscopy were used to examine the organization of DNA sequences in the Basidiomycete Schizophyllum commune. Short-period interspersion of repetitive and unique sequences is absent from the DNA of this wood-rotting fungus. Instead, repetitive sequences are found predominantly in several, perhaps 16, clusters averaging 225 kilobases in length. The results of this study, the first concerning a fungus in the important fungal Class Basidiomycetidae, are discussed in relation to: sequence organization in three other species of true fungi, fungal evolution and the regulation of gene expression.     

Genome size and complexity of the obligate fungal pathogen,Bremia lactucae

The size and organization of the genome of Bremia lactucae, a highly specialized fungal pathogen of lettuce, has been characterized using dot blot genomic reconstructions, reverse genomic blots, and genomic DNA reassociation kinetics. The haploid genome contains 5 × 10⁷ bp of DNA and 65% of the nuclear DNA is repeated. Low copy sequences are interspersed with repeated sequences in a short-period interspersion pattern. This pattern of genome organization is different to that described for other fungi. Although most fungi have been shown to contain some form of repetitive DNA other than the ribosomal repeat, the high percentage of repetitive DNA and the interspersion of low copy and repeated sequences are atypical of fungi characterized previously.     

Comparison of poly(A)-containing RNAs in different cell types of the lower eukaryote Schizophyllum commune

Poly(A)-containing RNAs were isolated from morphologically different cells of the fungus Schizophyllum commune. Using mRNA markers the number-average length of poly(A)-containing RNA in total RNA and in purified poly(A)-containing RNA was estimated as 1100 nucleotides. Number-average length of poly(A)-tracts was 33 nucleotides. 2.5% of total RNA is poly(A)-containing RNA and probably up to 7.5% are non-polyadenylated polydisperse RNA sequences. Saturation hybridization of poly(A)-containing RNA to gap-translated [3H]DNA resulted in 16% of the reactive single-copy DNA to become S1 nuclease resistant. It was found that purified poly(A)-containing RNA represented the entire RNA complexity, i.e. 10 000 different RNA sequences in S. commune. RNA sequences isolated from morphologically different mycelia and from fruiting and non-fruiting mycelia were identical for at least 90%.     

Characterization of the genome of Arabidopsis thaliana

The small crucifer Arabidopsis thaliana has many useful features as an experimental organism for the study of plant molecular biology. It has a four-week life-cycle, only five chromosomes and a genome size less than half that of Drosophila. To characterize the DNA sequence organization of this plant, we have randomly selected 50 recombinant lambda clones containing inserts with an average length of 12,800 base-pairs and analyzed their content of repetitive and unique DNA by various genome blot, restriction digestion and RNA blot procedures. The following conclusions can be drawn. The DNA represented in this random sample is composed predominantly of single-copy sequences. This presumably reflects the organization of the Arabidopsis genome as a whole and supports prior conclusions reached on the basis of kinetics of DNA reassociation. The DNA that encodes the ribosomal RNAs constitutes the only major class of cloned nuclear repetitive DNA. It consists of approximately 570 tandem copies of a heterogeneous 9900-base-pair repeat unit. There is an average of approximately 660 copies of the chloroplast genome per cell. Therefore, the chloroplast genome constitutes the major component of the repetitive sequences found in A. thaliana DNA made from whole plants. The inner cytosine residue in the sequence C-C-G-G is methylated more often than the outer in the tandem ribosomal DNA units, whereas very few differences in the methylation state of these two cytosine residues are detected in unique sequences.     

Polymorphism at the ribosomal DNA spacers and its relation to breeding structure of the widespread mushroom Schizophyllum commune

The common split-gilled mushroom Schizophyllum commune is found throughout the world on woody substrates. This study addresses the dispersal and population structure of this fungal species by studying the phylogeny and evolutionary dynamics of ribosomal DNA (rDNA) spacer regions. Extensive sampling (n = 195) of sequences of the intergenic spacer region (IGS1) revealed a large number of unique haplotypes (n = 143). The phylogeny of these IGS1 sequences revealed strong geographic patterns and supported three evolutionarily distinct lineages within the global population. The same three geographic lineages were found in phylogenetic analysis of both other rDNA spacer regions (IGS2 and ITS). However, nested clade analysis of the IGS1 phylogeny suggested the population structure of S. commune has undergone recent changes, such as a long distance colonization of western North America from Europe as well as a recent range expansion in the Caribbean. Among all spacer regions, variation in length and nucleotide sequence was observed between but not within the tandem rDNA repeats (arrays). This pattern is consistent with strong within-array and weak among-array homogenizing forces. We present evidence for the suppression of recombination between rDNA arrays on homologous chromosomes that may account for this pattern of concerted evolution.     

A chromosome 5-specific repetitive DNA sequence in rice (Oryza sativa L)

Repetitive DNA sequences in the rice genome comprise more than half of the nuclear DNA. The isolation and characterization of these repetitive DNA sequences should lead to a better understanding of rice chromosome structure and genome organization. We report here the characterization and chromosome localization of a chromosome 5-specific repetitive DNA sequence. This repetitive DNA sequence was estimated to have at least 900 copies. DNA sequence analysis of three genomic clones which contain the repeat unit indicated that the DNA sequences have two sub-repeat units of 37 bp and 19 bp, connected by 30-to 90-bp short sequences with high similarity. RFLP mapping and physical mapping by fluorescence in situ hybridization (FISH) indicated that almost all copies of the repetitive DNA sequence are located in the centromeric heterochromatic region of the long arm of chromosome 5. The strategy for cloning such repetitive DNA sequences and their uses in rice genome research are discussed.     

Two-dimensional gel analysis of repetitive nuclear DNA sequences in the genome of Physarum polycephalum. Developmental regulation of the ribosomal gene number

The composition of repetitive sequences in restriction patterns of nuclear DNA of Physarum polycephalum was determined by high-resolution gel analysis. Three types of repeated DNA fragments in the size range of (0.2-2) X 10(3) base pairs could be identified as discrete spots on the gels and distinguished by their abundance and above-average base composition of either guanine and cytosine (G + C) or adenine and thymidine (A + T). On comparing the DNA composition from exponentially growing plasmodia with that of starved plasmodia, which have become competent to sporulate and have lost 80% of their nuclei, no change was detected among the (A + T)-rich repeat fractions, whereas several of the (G + C)-rich fractions revealed fewer copies in the DNA prepared from starved cells. As shown by hybridization under saturating conditions, the reduction of several (G + C)-rich repeated sequences in the restricted nuclear DNA in sporulation-competent cells can be explained by a 64% elimination of the extrachromosomal nucleolar ribosomal DNA sequences.     

裂褶菌培养基形态学观察和DNA序列分析

目的通过观察裂褶菌在5种培养基上的生长状态、扫描电镜及DNA序列分析,了解该菌形态学及分子生物学等方面的特征。方法菌落转种于沙氏培养基(SDA),麦芽浸膏琼脂(MEA),马铃薯葡萄糖琼脂(PDA),玉米粉琼脂(CMA)和察氏琼脂(CZA)平皿培养基,27℃和37℃培养2周,观察菌落生长情况,进行扫描电镜检测及DNA序列分析。结果菌落在SDA,MEA和PDA上生长状态较好,呈蓬松白色羊毛状;尿素酶试验阳性,放线菌酮耐受试验阴性。光镜下见分支分隔菌丝、侧生的钉状突起及类水母体变异子实体。扫描电镜见菌丝分隔处闭锁联合、侧生钉状突起和泪滴状球形分泌物。经26S rDNAD1/D2区序列分析证实该菌株为裂褶菌。结论裂褶菌只有丝状型一种菌落形态;分支分隔菌丝及分隔处闭锁联合,侧生钉状突起和泪滴状球形分泌为其形态学特征;孢子由类水母状子实体产生。     

Characterization of the nuclear genome of pearl millet.

The nuclear genome of pearl millet has been characterized with respect to its size, buoyant density in CsCl equilibrium density gradients, melting temperature, reassociation kinetics and sequence organization. The genome size is 0.22 pg. The mol percent G + C of the DNA is calculated from the buoyant density and the melting temperature to be 44.9 and 49.7%, respectively. The reassociation kinetics of fragments of DNA 300 nucleotides long reveals three components: a rapidly renaturing fraction composed of highly repeated and/or foldback DNA, middle repetitive DNA and single copy DNA. The single copy DNA consists of 17% of the genome. 80% of the repetitive sequences are at least 5000 nucleotide pairs in length. Thermal denaturation profiles of the repetitive DNA sequences show high Tm values implying a high degree of sequence homogeneity. About half of the single copy DNA is short (750--1400 nucleotide paris) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotide pairs.     

Genetic transformation of Trametes versicolor to phleomycin resistance with the dominant selectable marker shble

We have developed a stable, DNA-mediated transformation system for the white-rot basidiomycete Trametes versicolor based on the dominant selectable marker shble (phleomycin resistance). We employed a vector containing the selectable marker under control of expression sequences from the basidiomycete Schizophyllum commune and a polyethylene glycol/ CaCl2 protoplast-fusion technique to introduce the transforming DNA. This transformation system generated stable phleomycin-resistant transformants at a frequency of four to seven transformants/microg of transforming DNA.     

A small nuclear precursor of messenger RNA in the cellular slime mold Dictyostelium discoideum

Previous work (Firtel et al., 1972) showed that messenger RNA from the cellular slime mold Dictyostelium discoideum, like that from mammalian cells, contains a sequence of about 100 adenylic acid residues at the 3′ end. We show here that Dictyostelium nuclei, labeled under a variety of conditions, do not contain material analogous to the large nuclear heterogeneous RNA found in mammalian cells. Rather, the majority of pulse-labeled nuclear RNA that is not a precursor of ribosomal RNA does contain at least one sequence of polyadenylic acid; this RNA, with an average molecular weight of 500,000, appears to be only 20% larger than cytoplasmic messenger RNA.Pulse-labeling experiments show that the nuclear poly(A)-containing RNA is a material precursor of messenger RNA. Whereas previous work showed that over 90% of messenger RNA sequences are transcribed from non-reiterated DNA, we show here that about 25% of nuclear poly (A)-containing RNA is transcribed from reiterated DNA sequences and only 75% from single-copy DNA. We present evidence that a large fraction of the nuclear poly(A)-containing RNA contains, at the 5′ end, a sequence of about 300 nucleotides that is transcribed from repetitive DNA, and which is lost before transport of messenger RNA into the cytoplasm.Based on these and other results, we present a model of arrangement of repetitive and single-copy DNA sequences in the Dictyostelium chromosome.     

Rearrangement of repeated DNA sequences during development of macronucleus in Tetrahymena thermophila.

Three clones of non-repetitive sequences and six clones containing repetitive sequences were obtained from micronuclear DNA of Tetrahymena thermophila. All the non-repetitive and three repetitive sequences had the same organization in micro- and macronuclear DNAs as revealed by blot hybridization. On the other hand, the remaining three clones with repetitive sequences had apparently different organization in the two nuclear DNAs. All these repetitive sequences showed a smear on the blot in addition to a number of discrete bands when micronuclear DNA was digested with EcoR I. In macronuclear DNAs, these sequences invariably became one or two bands and the smear disappeared. We conclude that, when a macronucleus develops from a micronucleus, the non-repetitive sequences amplify by more than 20 times with relatively few rearrangement, whereas some selected portions of repeated and/or repeat-contiguous sequences are amplified with rather extensive reorganization.     

Isolation and characterization of the highly repeated fraction of the banana genome

Although the nuclear genome of banana (Musa spp.) is relatively small (1C approximately 610 Mbp for M. acuminata), the results obtained from other sequenced genomes suggest that more than half of the banana genome may be composed of repetitive and non-coding DNA sequences. Knowledge of repetitive DNA can facilitate mapping of important traits, phylogenetic studies, BAC-based physical mapping, and genome sequencing/annotation. However, only a few repetitive DNA sequences have been characterized in banana. In this work, we used DNA reassociation kinetics to isolate the highly repeated fraction of the banana genome (M. acuminata 'Calcutta 4'). Two libraries, one prepared from Cot 相似文献   

CHARACTERIZATION OF THE NUCLEAR DNA OF HORDEUM VULGARE ROOT HAIRS: AMPLIFICATION DISAPPEARS UNDER SALT STRESS

In Hordeum vulgare L., the nucleus of differentiating root hairs contains amplified, extrachromosomal DNA sequences. Cytophotometry shows that the nuclei of trichoblasts and root hairs grown under normal conditions contain up to 50% more DNA than those grown in 200 mM salt. Although the root hairs develop and differentiate under salt stress, amplification of their nuclear DNA is suppressed. From this, we conclude that amplification is not necessary for differentiation at the cellular level. Characterization of the amplified nuclear DNA of the root hair is based on the physical/chemical nature of the DNA. The amplified sequences separate as a satellite band when total nucleic acids are centrifuged on CsCl gradients. Enzyme restriction of the satellite and main bands with Msp I and Hpa II followed by agarose gel electrophoresis shows that the satellite band is not more highly methylated than the main band. Restriction of the root hair DNA with EcoRI reveals repetitive DNA sequences not seen in similarly restricted whole root, leaf or salt-stressed root hair preparations. While these unique, repetitive sequences in the 2–6 kb region of the gel do not hybridize with ribosomal, chloroplast, or mitochondrial DNAs, RNA hybridization shows that some of them are transcribed. We believe that the amplified sequences are extrachromosomal based on their selective degradation during root hair senescence, their separation as a satellite band and their restriction patterns.