An analysis of the microsporidian genus Brachiola, with comparisons of human and insect isolates of Brachiola algerae

The genus Brachiola is the newest microsporidian genus established for a human infection with the type species being B. vesicularum in skeletal muscle. Subsequently, the microsporidium, Nosema algerae, identified from mosquitoes, was added to this genus because of morphological and physiological similarities. The present report illustrates a confirmed case of Brachiola algerae infecting skeletal muscle in a 56-year-old woman who was being treated for rheumatoid arthritis with immunosuppressive drugs. In the following study, these two human-infecting microsporidian species are ultrastructurally compared from human biopsy tissue. Additionally, Brachiola algerae from mosquitoes as reference B. algerae, was grown in athymic mice and compared to the human isolate in vivo, and in culture. B. algerae is morphologically identical in the host situations presented and different from B. vesicularum in human skeletal muscle. B. algerae has a consistently, slightly longer spore that typically contains one row of polar filament coils, while B. vesicularum typically contains two rows of polar filament coils and occasionally, one or three rows. In proliferative development, B. vesicularum forms protoplasmic extensions which do not occur on B. algerae, nor have they been reported on any other microsporidium. This report demonstrates that B. vesicularum and B. algerae are two different species of Brachiola that infect human skeletal muscle.     

The malaria genome sequencing project: complete sequence of Plasmodium falciparum chromosome 2

An international consortium has been formed to sequence the entire genome of the human malaria parasite Plasmodium falciparum. We sequenced chromosome 2 of clone 3D7 using a shotgun sequencing strategy. Chromosome 2 is 947 kb in length, has a base composition of 80.2% A + T, and contains 210 predicted genes. In comparison to the Saccharomyces cerevisiae genome, chromosome 2 has a lower gene density, a greater proportion of genes containing introns, and nearly twice as many proteins containing predicted non-globular domains. A group of putative surface proteins was identified, rifins, which are encoded by a gene family comprising up to 7% of the protein-encoding gene in the genome. The rifins exhibit considerable sequence diversity and may play an important role in antigenic variation. Sixteen genes encoded on chromosome 2 showed signs of a plastid or mitochondrial origin, including several genes involved in fatty acid biosynthesis. Completion of the chromosome 2 sequence demonstrated that the A + T-rich genome of P. falciparum can be sequenced by the shotgun approach. Within 2-3 years, the sequence of almost all P. falciparum genes will have been determined, paving the way for genetic, biochemical, and immunological research aimed at developing new drugs and vaccines against malaria.     

Molecular combing in studies of the genome organization and DNA replication

Molecular combing (MC) yields preparations where individual DNA molecules are uniformly stretched and are parallel to each other. Fluorescence in situ hybridization on such preparations allows an exact mapping of DNA sequences, and pulsed incorporation of halogenated deoxyuridine analogs and their detection using fluorochrome-conjugated antibodies makes it possible to visualize replication. The MC technique was adapted for studying DNA replication in isolated Drosophila melanogaster organs, and it was checked whether a mutation of the Suppressor of UnderReplication (SuUR) gene directly affected the replication fork rate.     

Genomic organization of the ribosomal DNA of sorghum and its close relatives

Summary The structure and organization of the ribosomal DNA (rDNA) of sorghum (Sorghum bicolor) and several closely related grasses were determined by gel blot hybridization to cloned maize rDNA. Monocots of the genus Sorghum (sorghum, shattercane, Sudangrass, and Johnsongrass) and the genus Saccharum (sugarcane species) were observed to organize their rDNA as direct tandem repeats of several thousand rDNA monomer units. For the eight restriction enzymes and 14 cleavage sites examined, no variations were seen within all of the S. bicolor races and other Sorghum species investigated. Sorghum, maize, and sugarcane were observed to have very similar rDNA monomer sizes and restriction maps, befitting their close common ancestry. The restriction site variability seen between these three genera demonstrated that sorghum and sugarcane are more closely related to each other than either is to maize. Variation in rDNA monomer lengths were observed frequently within the Sorghum genus. These size variations were localized to the intergenic spacer region of the rDNA monomer. Unlike many maize inbreds, all inbred Sorghum diploids were found to contain only one rDNA monomer size in an individual plant. These results are discussed in light of the comparative timing, rates, and modes of evolutionary events in Sorghum and other grasses. Spacer size variation was found to provide a highly sensitive assay for the genetic contribution of different S. bicolor races and other Sorghum species to a Sorghum population.     

Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species

Genomes of 11 Quercus species were characterized using cytogenetic (Giemsa C-banding, fluorochrome banding), molecular-cytogenetic (fluorescence in situ hybridization, FISH, to ribosomal genes) and molecular (dot-blot for ribosomal gene-copy number assessment) techniques. Ribosomal genes are the first DNA sequences to be physically mapped in oaks, and the copy number of the 18S-5.8S-26 S rRNA genes is estimated for the first time. Oak karyotypes were analysed on the basis of DAPI banding and FISH patterns; five marker chromosomes were found. In addition, chromosomal organization of ribosomal genes with respect to AT- and GC-differentiated heterochromatin was studied. Fluorochrome staining produced very similar CMA/DAPI banding patterns, and the position and number of ribosomal loci were identical for all the species studied. The 18S-5.8S-26 S rRNA genes in oak complements were represented by a major locus at the subterminal secondary constriction (SC) of the only subtelocentric chromosome pair and a minor locus at paracentromeric SC of one metacentric pair. The only 5 S rDNA locus was revealed at the paracentromeric region of the second largest metacentric pair. A striking karyotypic similarity, shown by both fluorochrome banding and FISH patterns, implies close genome relationships among oak species no matter their geographic origin (European or American) or their ecophysiology (deciduous or evergreens). Dot-blot analysis gave preliminary evidence for different copy numbers of 18S-5.8S-26 S rRNA genes in diploid genomes of Q. cerris, Q. ilex, Q. petraea, Q. pubescens and Q. robur (2700, 1300, 2200, 4000 and 2200 copies, respectively) that was correlated with the size polymorphism of the major locus. Received: 26 February 1999 / Accepted: 16 March 1999     

The complete nucleotide sequence and its organization of the genome of Barley yellow dwarf virus-GAV

The complete nucleotide sequence of genomic RNA of BYDV-GAV was determined. It comprised 5685 nucleotides and contained six open reading frames and four un-translated regions. The size and organization of BYDV-GAV genome were similar to those of BYDV PAV-aus. The nucleotide and deduced amino acid sequences of the six ORFs were aligned and compared with those of other luteoviruses. The results showed that there was a high degree of identity between BYDV-GAV and MAV-PS1 in all ORFs except ORF5 and ORF6, which had only 87.4% and 70.2% identities respectively. The reported genomic nucleotide sequence of MAV was shorter than that of BYDV-GAV, but the comparison of the genomic nucleotide sequences for MAV-PS1 and GAV showed 90.4% sequence identity for the same region of the genome. According to the level of sequence similarities, BYDV-GAV should be closely related to BYDV-MAV.     

The complete nucleotide sequence and its organization of the genome of Barley yellow dwarf virus-GAV

The Barley yellow dwarf disease (BYD) was firstly recognized as an aphid transmitted virus disease by Oswald and Houston[1] in 1951. Now, Barley yel-low dwarf viruses (BYDVs) belong to members of the plant virus family Luteoviridae. They are phloem- limited and obligately transmitted in the circula-tive/persistent manner by several species of cereal aphids and can cause significant economic losses worldwide because of damage to barley, wheat, and oats. In China, BYDVs cause mainly yello…     

The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome

Eukaryotic ribosomes are made of two components, four ribosomal RNAs, and approximately 80 ribosomal proteins (r-proteins). The exact number of r-proteins and r-protein genes in higher plants is not known. The strong conservation in eukaryotic r-protein primary sequence allowed us to use the well-characterized rat (Rattus norvegicus) r-protein set to identify orthologues on the five haploid chromosomes of Arabidopsis. By use of the numerous expressed sequence tag (EST) accessions and the complete genomic sequence of this species, we identified 249 genes (including some pseudogenes) corresponding to 80 (32 small subunit and 48 large subunit) cytoplasmic r-protein types. None of the r-protein genes are single copy and most are encoded by three or four expressed genes, indicative of the internal duplication of the Arabidopsis genome. The r-proteins are distributed throughout the genome. Inspection of genes in the vicinity of r-protein gene family members confirms extensive duplications of large chromosome fragments and sheds light on the evolutionary history of the Arabidopsis genome. Examination of large duplicated regions indicated that a significant fraction of the r-protein genes have been either lost from one of the duplicated fragments or inserted after the initial duplication event. Only 52 r-protein genes lack a matching EST accession, and 19 of these contain incomplete open reading frames, confirming that most genes are expressed. Assessment of cognate EST numbers suggests that r-protein gene family members are differentially expressed.     

The mitochondrial DNA of Dictyostelium discoideum: complete sequence, gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564 bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4 kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii. Received: 5 July 1999 / Accepted: 17 January 2000     

Automated DNA sequencing and analysis of the human genome

In the past few years, striking advances have been made in automating DNA sequence analysis. Currently, efforts are underway to automate and improve DNA purification, mapping, and data processing procedures. The predictable advances in these technologies should soon place us in a position to sequence the entire human genome. The information derived from this project will have profound implications for basic biology and clinical medicine alike.     

DNA sequence organization in the genome of Cycas revoluta

The pattern of DNA sequence organization in the genome of Cycas revoluta was analyzed by DNA/DNA reassociation. Reassociation of 400 base pair (bp) fragments to various C₀t values indicates the presence of at least four kinetic classes: the foldback plus very highly repetitive sequences (15%), the fast repeats (24%), the slow repeats (44%), and the single copy (17%). The latter component reassociates with a rate constant 1×10^–4 M^–1S^–1 corresponding to a complexity of 1.6× 10⁶ kb per haploid genome. A haploid C. revoluta nucleus contains approximately 10.3 pg DNA. The single-copy sequences account for about 28% of the DNA, but only 17% reassociate with single-copy kinetics because of interspersion with repetitive sequences. — The interspersion of repetitive and single-copy sequences was examined by reassociation of DNA fragments of varying length to C₀t values of 70 and 500. A major (65%) and homogeneous class of single-copy sequences averaging 1,100 bp in length is interspersed in a short period pattern with repeated sequences. A minor (35%) heterogeneous single-copy component is interspersed in a long-period pattern. The majority of repetitive sequences have a length distribution of 100–350 bp with subclasses averaging 150 and 300 bp in length. Repeat sequences with a wide range in sizes exceeding 2 kilobase pair (kb) are also present in this genome. — The size and distribution of inverted repeat (ir) sequences in the DNA of C. revoluta were studied by electron microscopy. It is estimated that there are approximately 4 × 10⁶ ir pairs (one per 2.33 kb) that form almost equal numbers of looped and unlooped palindromes. This high value is 2.5 times that found in wheat DNA. These palindromes are in general randomly distributed in the genome with an average interpalindrome distance of 1.6 kb. The majority (about 85%) of ir sequences of both types of palindromes belong to a main-size class, with an average length of 210 bp in the unlooped and and 163 bp in the looped type. These values are comparable to those reported for some other plant and animal genomes. Distribution of length of single stranded loops showed a main-size class (75%) with an average length of 220 bp.     

The organization of DNA sequences in the mouse genome

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag⁺-Cs₂SO₄ density gradient centrifugation. — According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2×10⁴ copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).— Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides. —Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides. — The organization of mouse genome analyzed by Ag⁺-Cs₂SO₄ density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.     

Automated DNA sequencing and the analysis of the human genome

The Human Genome Initiative is a complex, multifaceted, international effort to establish a massive data base of map and sequence information for humans and other organisms. The success of this initiative is dependent upon the development of new technologies for the analysis of genomes. In this paper, an overview of the Human Genome Initiative is presented, and the current status of efforts to automate large-scale DNA sequence analysis is reviewed.     

DNA sequence organization and transcription of the chicken genome

A new approach has been used to examine DNA sequence organization in the chicken genome. The interspersion pattern was determined by studying the fraction of labelled DNA fragments of different lengths that hybridized to an excess of short chicken repeated DNA sequences. The results indicate that chicken DNA has a pattern of sequence organization quite different than the standard ‘Xenopus’ or ‘Drosophila’ patterns. Two classes of unique sequences are found. One, 34% of the genome, consists of unique sequences approx. 4 kb long interspersed with repeated sequences. The second, non-interspersed fraction, 38% of the genome, consists of unique sequences found in long tracts, a minimum of approx. 22 kb in length. In an attempt to determine whether a relationship exists between DNA sequence organization and the distribution of structural genes we have isolated chicken DNA sequences belonging to different interspersion classes and tested each for the presence of structural genes by hybridization to excess poly(A)⁺ mRNA. Sequences complementary to poly(A)⁺ mRNA can be found with approximately the same frequency in both the non-interspersed fraction of the genome and a repeat-contiguous fraction enriched for interspersed sequences.