A chicken middle-repetitive DNA sequence which shares homology with mammalian ubiquitous repeats.

We have identified and sequenced two members of a chicken middle repetitive DNA sequence family. By reassociation kinetics, members of this family (termed CRl) are estimated to be present in 1500-7000 copies per chicken haploid genome. The first family member sequenced (CRlUla) is located approximately 2 kb upstream from the previously cloned chicken Ul RNA gene. The second CRl sequence (CRl)Va) is located approximately 12 kb downstream from the 3' end of the chicken ovalbumin gene. The region of homology between these two sequences extends over a region of approximately 160 base pairs. In each case, the 160 base pair region is flanked by imperfect, but homologous, short direct repeats 10-15 base pairs in length. When the CRl sequences are compared with mammalian ubiquitous interspersed repetitive DNA sequences (human Alu and Mouse Bl families), several regions of extensive homology are evident. In addition, the short nucleotide sequence CAGCCTGG which is completely conserved in ubiquitous repetitive sequence families from several mammalian species is also conserved at a homologous position in the chicken sequences. These data imply that at least certain aspects of the sequence and structure of these interspersed repeats must predate the avian-mammalian divergence. It seems that the CRl family may possibly represent an avian counterpart of the mammalian ubiquitous repeats.     

Assessment of the base sequence homology between the two subtypes of equine herpesvirus 1.

The magnitude of the genetic relatedness of the two antigenic subtypes of equine herpesvirus 1 (EHV-1) was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA from one EHV-1 subtype was allowed to reassociate in the presence or absence of the unlabeled heterologous viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating 10 to 20% homology between the two EHV-1 genomes. Similar estimates of the amount of homology between the genomes of the two EHV-1 subtypes were obtained by determining the maximum fraction of labeled viral DNA that could be made resistant to S1 nuclease by hybridization with a large molar excess of the unlabeled, heterologous viral DNA. Analysis of the thermal stability of the subtype 1-subtype 2 heteroduplex DNA indicated approximately 30% base pair mismatching within the hybrid DNA molecules. Cross-hybridization of 32P-labeled virion DNA to nitrocellulose blots of restriction endonuclease cleavage fragments of each EHV-1 subtype DNA indicated that the observed homology between the two viruses was nonuniformly distributed with the viral genome. No homology could be detected between the DNA of either EHV-1 subtype and that of a strain of equine cytomegalovirus (EHV-2). The data suggest that the two biotypes of EHV-1 have arisen by divergent evolution from a common progenitor herpesvirus.     

The DNA sequence of the human cytomegalovirus genome.

In the first part of this article we review what has been learnt from the analysis of the sequence of HCMV. A summary of this information is presented in the form of an updated map of the viral genome. HCMV is representative of a major lineage of herpesviruses distinct from previously sequenced members of this viral family and demonstrates striking differences in genetic content and organization. The virus encodes approximately 200 genes, including nine gene families, a large number of glycoprotein genes, and homologues of the human HLA class I and G protein-coupled receptor genes. The HCMV sequence thus provides a sound basis for future molecular studies of this highly complex eukaryotic virus. The second part discusses the practical rate of DNA sequencing as deduced from this and other studies. The 229 kilobase pair DNA genome of human cytomegalovirus (HCMV) strain AD169 is the largest contiguous sequence determined to date, and as such provides a realistic benchmark for assessing the practical rate of DNA sequencing as opposed to theoretical calculations which are usually much greater. The sequence was determined manually and we assess the impact of new developments in DNA sequencing.     

Method for determination of nucleotide sequence homology between viral genomes by DNA reassociation kinetics.

A model and appropriate equations were derived for the quantitative estimation of nucleotide sequence homology between two partially related viral genomes by measurement of the initial rate of reassociation of one labeled DNA in the presence of a second unlabeled DNA. The validity and usefulness of this procedure were demonstrated by the analysis of the reassociation kinetics of labeled adenovirus 7 DNA in the presence of unlabeled adenovirus 2 DNA. Based on DNA reassociation, the extent of homology between adenovirus 2 and 7 genomes was found to be 10 to 12%. The duplex formed between adenovirus 2 and 7 DNA had the appropriate thermal stability for a well-matched DNA-DNA hybrid.     

DNA sequence repetition in the genome of the American oyster.

The genome of Crassostrea virignica, the American oyster, has been studied by reassociation kinetics in order to construct a profile of DNA sequence frequency components. Oyster DNA has been shown to contain at least 51% single copy DNA sequences and two classes of middle repetitive DNA. The major repetitive class contains sequences which are repeated on the average 20 times and comprise 29% of oyster DNA. The other class represents 10% of oyster DNA and contains sequences repeated approx. 3000 times. In addition the DNA of oyster contains at least 1% foldback sequences. The spectrum of DNA repetition components in the American oyster is similar to that found in the genomes of other mollusks.     

A unique repetitive DNA sequence in the Myxococcus xanthus genome.

We found a novel type of repetitive DNA sequence in the Myxococcus xanthus genome. The first repetitive sequence is located in the spacer region between the ops and tps genes. We cloned five other repetitive sequences using the first repetitive sequence as a probe and determined their nucleotide sequences. Comparison of these sequences revealed that the repetitive sequences consist of a 87-bp core sequence and that some clones share additional homology on their flanking regions.     

Comparison of the sequence specificity of bleomycin cleavage in two slightly different DNA sequences.

The sequence specificity of bleomycin damage was investigated utilising 340 bp alpha-DNA (a middle repetitive sequence in the human genome) as a target sequence. The following significant facts were found:- i) The dinucleotides GT and GC were cleaved on all occasions, GA most of the time, and AT, AC, GG and AA cleaved some of the time; ii) The base immediately 5' to the purine-pyrimidine dinucleotides was found to be statistically highly significant in determining the degree of damage caused by bleomycin, while other nearest neighbour bases had no significant effect; iii) The sequence specificity of bleomycin damage was determined on both strands and it was found that damage on either strand follows the above dinucleotide preference and is independent of the extent of damage on the opposite strand; iv) Bleomycin damage was compared between genomic 340 bp alpha-DNA and a cloned alpha-DNA with eleven base substitutions relative to the "consensus" sequence. There were forty-nine detectable differences in intensity of damage between these two DNA molecules. Although four of the differences can be directly attributed to changes in base sequence, the remaining differences were not at the base substitution sites. Some of the differences were over fifty base pairs from the nearest base substitution. We propose that the majority of these differences are due to microvariation in the structure of DNA with a slightly different DNA sequence.     

DNA Data Bank of Japan at work on genome sequence data.

We at the DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp) have recently begun receiving, processing and releasing EST and genome sequence data submitted by various Japanese genome projects. The data include those for human, Arabidopsis thaliana, rice, nematode, Synechocystis sp. and Escherichia coli. Since the quantity of data is very large, we organized teams to conduct preliminary discussions with project teams about data submission and handling for release to the public. We also developed a mass submission tool to cope with a large quantity of data. In addition, to provide genome data on WWW, we developed a genome information system using Java. This system (http://mol.genes.nig.ac.jp/ecoli/) can in theory be used for any genome sequence data. These activities will facilitate processing of large quantities of EST and genome data.     

DNA sequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species.

DNA hybridization experiments indicate that the genome of a tumorigenic poxvirus. Shope fibroma virus (SFV), possesses sequence homology with DNA isolated from uninfected rabbit cells. Southern blotting experiments, either with high-complexity rabbit DNA as probe and SFV restriction fragments as targets or with high-specific activity, 32P-labeled, cloned SFV sequences as probes and rabbit DNA as target, indicate that the homologous sequences map at two locations within the viral genome, one in each copy of the terminal inverted repeat sequences. Unexpectedly, Southern blots revealed that the homologous host sequences reside in a rabbit extrachromosomal DNA element. This autonomous low-molecular-weight DNA species could be specifically amplified by cycloheximide treatment and was shown by isopycnic centrifugation in cesium chloride-ethidium bromide to consist predominantly of covalently closed circular DNA molecules. DNA sequencing of pSIC-9, a cloned 1.9-kilobase fragment of the rabbit plasmid species, indicated extensive homology at the nucleotide level over a 1.5-kilobase stretch of the viral terminal inverted repeat. Analysis of open reading frames in both the plasmid and SFV DNA revealed that (i) the N-terminal 157-amino acid sequence of a potential 514-amino acid SFV polypeptide is identical to the N-terminal 157 amino acids of one pSIC-9 open reading frame, and (ii) a second long pSIC-9 open reading frame of 361 amino acids, although significantly diverged from the comparable nucleotide sequence in the virus, possessed considerable homology to a family of cellular protease inhibitors, including alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. The potential role of such cellular plasmid-like DNA species as a mediator in the exchange of genetic information between the host cell and a cytoplasmically replicating poxvirus is discussed.     

An RFLP species-specific DNA sequence for the A genome of rice.

A DNA sequence, pOs6.20, was cloned from the nuclear genome of cultivated rice (Oryza sativa L.) by hybridization with a human minisatellite sequence. At high hybridization stringency, a subfragment of the rice sequence, pOs6.20.3, detected low-copy restriction fragment length polymorphisms (RFLPs), which behaved as Mendelian genetic markers. This subfragment detected multicopy RFLPs between an indica and a javanica cultivar at medium hybridization stringency. The sequences detected by pOs6.20.3 at high hybridization stringency did not occur in rice species possessing the B, C, D, E, and F genomes. These results suggest that the RFLPs detected by this sequence can be placed on a genetic map of rice and that this sequence can be used as a species-specific probe for the A genome of rice.     

Drosophila topoisomerase II double-strand DNA cleavage: analysis of DNA sequence homology at the cleavage site.

In order to study the sequence specificity of double-strand DNA cleavage by Drosophila topoisomerase II, we have mapped and sequenced 16 strong and 47 weak cleavage sites in the recombinant plasmid p pi 25.1. Analysis of the nucleotide and dinucleotide frequencies in the region near the site of phosphodiester bond breakage revealed a nonrandom distribution. The nucleotide frequencies observed would occur by chance with a probability less than 0.05. The consensus sequence we derived is 5'GT.A/TAY decrease ATT.AT..G 3', where a dot means no preferred nucleotide, Y is for pyrimidine, and the arrow shows the point of bond cleavage. On average, strong sites match the consensus better than weak sites.     

Histone genes of Volvox carteri: DNA sequence and organization of two H3-H4 gene loci.

Two Volvox genomic clones each containing a pair of histone H3-H4 genes were sequenced. In both loci the H3 and H4 genes show outwardly divergent polarity, their coding regions being separated by short intercistronic sequences containing TATA boxes and a conserved 14-bp element. The 3' untranslated regions contain a characteristic motif with hyphenated dyad symmetry otherwise only found associated with animal histone genes. Derived amino acid sequences of histones H3 and H4 are highly conserved and identical between the two sets. The Volvox H3 genes both contain one intron whose relative position is shifted by one basepair. Sequence comparisons led to a new interpretation of intron sliding. The Volvox H3 gene structure combines the exon-intron organization of fungal H3 and vertebrate H3.3 genes with a termination signal typical for animal H3.1 genes. These features are discussed in view of histone gene evolution.