Replication of the baculovirus genome

The review describes the current state of studying the baculovirus DNA replication. The structural organization of replication initiation sites and replication intermediates are considered. Attention is focused on virus replication factors, including DNA polymerase, helicase, IE-1, LEF-1, LEF-2, and LEF-3.     

Assembly errors cause false tandem duplicate regions in the chicken (Gallus gallus) genome sequence

The complexity of eukaryote genomes makes assembly errors inevitable in the process of constructing reference genomes. Next-generation sequencing (NGS) could provide an efficient way to validate previously assembled genomes. Here, we exploited NGS data to interrogate the chicken reference genome and identified 35 pairs of nearly identical regions with >99.5 % sequence similarity and a median size of 109 kb. Several lines of evidence, including read depth, the composition of junction sequences, and sequence similarity, suggest that these regions present genome assembly errors and should be excluded from forthcoming genomic studies.     

Replication fork reversal and the maintenance of genome stability

The progress of replication forks is often threatened in vivo, both by DNA damage and by proteins bound to the template. Blocked forks must somehow be restarted, and the original blockage cleared, in order to complete genome duplication, implying that blocked fork processing may be critical for genome stability. One possible pathway that might allow processing and restart of blocked forks, replication fork reversal, involves the unwinding of blocked forks to form four-stranded structures resembling Holliday junctions. This concept has gained increasing popularity recently based on the ability of such processing to explain many genetic observations, the detection of unwound fork structures in vivo and the identification of enzymes that have the capacity to catalyse fork regression in vitro. Here, we discuss the contexts in which fork regression might occur, the factors that may promote such a reaction and the possible roles of replication fork unwinding in normal DNA metabolism.     

Chlamydia: five years A.G. (after genome)

The obligate intracellular pathogens Chlamydiaceae are the agents of several human diseases. They cannot be genetically manipulated and they survive and replicate in a unique intracellular organelle called the inclusion. In the past five years, publications of the genome sequences of several strains have opened new areas of research. Some of these new advances are presented here.     

Replication of the rotavirus genome requires an active ubiquitin-proteasome system

Here we show that the ubiquitin-proteasome system is required for the efficient replication of rotavirus RRV in MA104 cells. The proteasome inhibitor MG132 decreased the yield of infectious virus under conditions where it severely reduces the synthesis of not only viral but also cellular proteins. Addition of nonessential amino acids to the cell medium restored both viral protein synthesis and cellular protein synthesis, but the production of progeny viruses was still inhibited. In medium supplemented with nonessential amino acids, we showed that MG132 does not affect rotavirus entry but inhibits the replication of the viral genome. It was also shown that it prevents the efficient incorporation into viroplasms of viral polymerase VP1 and the capsid proteins VP2 and VP6, which could explain the inhibitory effect of MG132 on genome replication and infectious virus yield. We also showed that ubiquitination is relevant for rotavirus replication since the yield of rotavirus progeny in cells carrying a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme was reduced at the restrictive temperature. In addition, overexpression of ubiquitin in MG132-treated MA104 cells partially reversed the effect of the inhibitor on virus yield. Altogether, these data suggest that the ubiquitin-proteasome (UP) system has a very complex interaction with the rotavirus life cycle, with both the ubiquitination and proteolytic activities of the system being relevant for virus replication.     

Replication Protein A (RPA): The Eukaryotic SSB

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is highly conserved in eukaryotes. RPA plays essential roles in many aspects of nucleic acid metabolism, including DNA replication, nucleotide excision repair, and homologous recombination. In this review, we provide a comprehensive overview of RPA structure and function and highlight the more recent developments in these areas. The last few years have seen major advances in our understanding of the mechanism of RPA binding to DNA, including the structural characterization of the primary DNA-binding domains (DBD) and the identification of two secondary DBDs. Moreover, evidence indicates that RPA utilizes a multistep pathway to bind single-stranded DNA involving a particular molecular polarity of RPA, a mechanism that is apparently used to facilitate origin denaturation. In addition to its mechanistic roles, RPA interacts with many key factors in nucleic acid metabolism, and we discuss the critical nature of many of these interactions to DNA metabolism. RPA is a phosphorylation target for DNA-dependent protein kinase (DNA-PK) and likely the ataxia telangiectasia-mutated gene (ATM) protein kinase, and recent observations are described that suggest that RPA phosphorylation plays a significant modulatory role in the cellular response to DNA damage.     

Replication process of the parvovirus H-1. IX. Physical mapping studies of the H-1 genome.

The cleavage map of H-1 replicative-form DNA to the bacterial restriction endonuclease EcoRI, HaeII, HaeIII, HindII, HindIII, and HpaII has been determined. The 5'-phosphoryl end of the viral strand is on the right end of the molecule at or near the replication origin. Evidence is presented for the presence of inverted self-complementary sequences at the right end that differ from those at the left end. These sequences allow a foldback of the DNA after denaturation, and a minority of the native replicative-form DNA has the foldback configuration. The possible role of these structures in H-1 DNA synthesis is discussed.     

Comparative physical mapping between Oryza sativa (AA genome type) and O. punctata (BB genome type)

A comparative physical map of the AA genome (Oryza sativa) and the BB genome (O. punctata) was constructed by aligning a physical map of O. punctata, deduced from 63,942 BAC end sequences (BESs) and 34,224 fingerprints, onto the O. sativa genome sequence. The level of conservation of each chromosome between the two species was determined by calculating a ratio of BES alignments. The alignment result suggests more divergence of intergenic and repeat regions in comparison to gene-rich regions. Further, this characteristic enabled localization of heterochromatic and euchromatic regions for each chromosome of both species. The alignment identified 16 locations containing expansions, contractions, inversions, and transpositions. By aligning 40% of the punctata BES on the map, 87% of the punctata FPC map covered 98% of the O. sativa genome sequence. The genome size of O. punctata was estimated to be 8% larger than that of O. sativa with individual chromosome differences of 1.5-16.5%. The sum of expansions and contractions observed in regions >500 kb were similar, suggesting that most of the contractions/expansions contributing to the genome size difference between the two species are small, thus preserving the macro-collinearity between these species, which diverged approximately 2 million years ago.     

Reproduction and Larval Morphology of Five Ophryotrocha Species (Poly cha eta, Dorvilleidae)

Reproduction and larval morphology of Ophryotrocha puerilis, O. labronica, O. nologlandulata, O. hartmanni , and O. bacci are described. All species have been cultivated in the laboratory. The morphology of adults and larvae is uniform, but there is a considerable variation in the shape of the egg masses. The interrelations between members of the genus Ophryotrocha are discussed: Gonochoric species have the same type of egg mass and the same chromosome number. O. puerilis is unique as a protandrous hermaphrodite. Among contemporary hermaphrodites with male and female segments, fusiform egg masses with a rigid surface are correlated with one larval type, gelatinous egg masses with another larval type.     

Poly(ADP-ribose) polymerases: managing genome stability

The importance of poly(ADP-ribose) metabolism in the maintenance of genomic integrity following genotoxic stress has long been firmly established. Poly(ADP-ribose) polymerase-1 (PARP-1) and its catabolic counterpart, poly(ADP-ribose) glycohydrolase (PARG) play major roles in the modulation of cell responses to genotoxic stress. Recent discoveries of a number of other enzymes with poly(ADP-ribose) polymerase activity have established poly(ADP-ribosyl)ation as a general biological mechanism in higher eukaryotic cells that not only promotes cellular recovery from genotoxic stress and eliminates severely damaged cells from the organism, but also ensures accurate transmission of genetic information during cell division. Additionally, emerging data suggest the involvement of poly(ADP-ribosyl)ation in the regulation of intracellular trafficking, memory formation and other cellular functions. In this brief review on PARP and PARG enzymes, emphasis is placed on PARP-1, the best understood member of the PARP family and on the relationship of poly(ADP-ribosyl)ation to cancer and other diseases of aging.     

Over-representation of the disease associated (CAG) and (CGG) repeats in the human genome.

Expansion of trimer repeats has recently been described as a new type of human mutation. Of the 64 possible trimer compositions, only the CGG and CAG repeats have been implicated in genetic diseases. This study intends to address two questions: (1) What makes the CGG and CAG repeats unique? (2) Could other trimer repeats be involved in this type of mutation? By computer analysis of trimer and hexamer frequency distributions in approximately 10 Mb of human DNA, twenty trimer motifs (ten complementary pairs) have been identified that are the most likely to be expanded. The frequency distribution study also indicated that the expanded trimer motif in Fragile-X syndrome is GGC instead of CGG. DNA linguistics studies revealed that the GGC/GCC and CAG/CTG repeats were over-represented in the human genome. Further analysis of base composition suggested that the CCA/TGG repeats may be involved in the trimer expansion mutation since they possessed many similar characteristics to GGC/GCC and CAG/CTG. The computer aided sequence analysis studies reported here may help to understand the molecular mechanisms of trimer repeat expansion.     

Complete sequence of the amphioxus (Branchiostoma lanceolatum) mitochondrial genome: relations to vertebrates.

The complete nucleotide sequence of the mitochondrial DNA of the amphioxus Branchiostoma lanceolatum has been determined. This mitochondrial genome is small (15 076 bp) because of the short size of the two rRNA genes and the tRNA genes. In addition, this genome contains a very short non-coding region (57 bp) with no sequence reminiscent of a control region. The organisation of the coding genes, as well as of the two rRNA genes, is identical to that of the sea lamprey. Some differences in the repartition of the tRNA genes occur when compared to the lamprey. The mitochondrial codon usage of the amphioxus is reminiscent of that of urochordates since the AGA codon is read as a glycine and not as a stop codon as in vertebrates. Moreover, the base composition at the wobble positions of the codon is strongly biased toward guanine. Altogether, these data clearly emphasise the close relationships between amphioxus and vertebrates, and reinforce the notion that prochordates may be viewed as the brother group of vertebrates.     

Replication of a mammalian genome: the role of de novo protein biosynthesis during S phase

ts14 is a temperature-sensitive Chinese hamster lung cell mutant that ceases protein biosynthesis within a short time of transfer to nonpermissive temperature (Haralson and Roufa, 1975; Roufa and Haralson, 1975; Roufa and Reed, 1975). This mutant contains a revertible, presumably a point mutation that renders its 60S ribosomal subunit thermolabile (Haralson and Roufa, 1975). In this report, we describe the relationship between the conditional ability of ts14 to synthesize protein during S phase and the replication of its DNA.After transfer to nonpermissive temperature (39°C), where ts14 synthesizes protein at a rate approximately 20 fold less than wild-type cells, synchronous cultures of the mutant performed all the processes required for replication of their DNA. During prolonged incubations at nonpermissive temperature, S phase ts14 completed approximately one round of DNA replication semi-conservatively as judged by density-transfer experiments. Pulse-labeling experiments performed on S phase cells revealed that ts14 synthesized the intermediates of discontinuous DNA replication at nonpermissive and permissive temperatures at similar rates. In these tests, the mutant was not substantially different from wild-type at both culture temperatures. At the nonpermissive temperature, however, ts14 synthesized significantly less nuclear protein (that is, histone) than did wild-type cells, and the mutant's chromatin appeared deficient in histone by virtue of its increased sensitivity to nuclease.     

Formation of the genome of the alligator gar Lepisosteus osseus (Ganoidomorpha) genome

Genome structure of the alligator gar was studied by means of a comparison of reassociation kinetics of short and long DNA fragments, an estimation of hyperchromicity of reassociated repetitive DNA as a function of fragments length, and length estimation of S1-resistant duplexes by gel filtration. It was shown that most of the repeated sequences in the alligator gar DNA are no less than 2000 b.p. long and weakly divergent. Little or no interspersion of unique and short repeated sequences were observed in this genome. No highly divergent repeats were found in the alligator gar genome.     

Rat Genome Database (RGD): mapping disease onto the genome

The Rat Genome Database (RGD, http://rgd.mcw.edu) is an NIH-funded project whose stated mission is ‘to collect, consolidate and integrate data generated from ongoing rat genetic and genomic research efforts and make these data widely available to the scientific community’. In a collaboration between the Bioinformatics Research Center at the Medical College of Wisconsin, the Jackson Laboratory and the National Center for Biotechnology Information, RGD has been created to meet these stated aims. The rat is uniquely suited to its role as a model of human disease and the primary focus of RGD is to aid researchers in their study of the rat and in applying their results to studies in a wider context. In support of this we have integrated a large amount of rat genetic and genomic resources in RGD and these are constantly being expanded through ongoing literature and bulk dataset curation. RGD version 2.0, released in June 2001, includes curated data on rat genes, quantitative trait loci (QTL), microsatellite markers and rat strains used in genetic and genomic research. VCMap, a dynamic sequence-based homology tool was introduced, and allows researchers of rat, mouse and human to view mapped genes and sequences and their locations in the other two organisms, an essential tool for comparative genomics. In addition, RGD provides tools for gene prediction, radiation hybrid mapping, polymorphic marker selection and more. Future developments will include the introduction of disease-based curation expanding the curated information to cover popular disease systems studied in the rat. This will be integrated with the emerging rat genomic sequence and annotation pipelines to provide a high-quality disease-centric resource, applicable to human and mouse via comparative tools such as VCMap. RGD has a defined community outreach focus with a Visiting Scientist program and the Rat Community Forum, a web-based forum for rat researchers and others interested in using the rat as an experimental model. Thus, RGD is not only a valuable resource for those working with the rat but also for researchers in other model organisms wishing to harness the existing genetic and physiological data available in the rat to complement their own work.