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Most cancer genomes are characterized by the gain or loss of copies of some sequences through deletion, amplification or unbalanced translocations. Delineating and quantifying these changes is important in understanding the initiation and progression of cancer, in identifying novel therapeutic targets, and in the diagnosis and prognosis of individual patients. Conventional methods for measuring copy-number are limited in their ability to analyse large numbers of loci, in their dynamic range and accuracy, or in their ability to analyse small or degraded samples. This latter limitation makes it difficult to access the wealth of fixed, archived material present in clinical collections, and also impairs our ability to analyse small numbers of selected cells from biopsies. Molecular copy-number counting (MCC), a digital PCR technique, has been used to delineate a non-reciprocal translocation using good quality DNA from a renal carcinoma cell line. We now demonstrate µMCC, an adaptation of MCC which allows the precise assessment of copy number variation over a significant dynamic range, in template DNA extracted from formalin-fixed paraffinembedded clinical biopsies. Further, µMCC can accurately measure copy number variation at multiple loci, even when applied to picogram quantities of grossly degraded DNA extracted after laser capture microdissection of fixed specimens. Finally, we demonstrate the power of µMCC to precisely interrogate cancer genomes, in a way not currently feasible with other methodologies, by defining the position of a junction between an amplified and nonamplified genomic segment in a bronchial carcinoma. This has tremendous potential for the exploitation of archived resources for high-resolution targeted cancer genomics and in the future for interrogating multiple loci in cancer diagnostics or prognostics. Copyright  2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Salmonella Genomic Island-1 (SGI-1) harbors a cluster of genes encoding multidrug resistance (MDR). SGI-1 is horizontally transmissible and is therefore of significant public health concern. This study presents two novel realtime PCRs detecting three SGI-1 protein-coding genes and a SGI-1 fingerprinting assay. These assays were applied to 445 European enterobacterial isolates. Results from real-time PCRs were comparable to those obtained from gelbased PCRs used for the detection of SGI-1, but were rapid to perform and suitable for large-scale screening. Furthermore, real-time PCRs also detected SGI-1 even when only part of the island was present in bacterial isolates. No trace of SGI-1 was detected in isolates other than Salmonella enterica. The fingerprints showed that regions of SGI-1 outside the MDR region exhibited genomic variations between isolates. In conclusion, the realtime PCRs described here are suitable for the detection of SGI-1 in bacterial isolates. Further studies are necessary to elucidate divergence in its non-MDR region.

Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa--protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies. Two species, C. hominis and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans, whereas C. parvum also infects other mammals. Here we describe the eight-chromosome approximately 9.2-million-base genome of C. hominis. The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.

The apicomplexan Cryptosporidium parvum is one of the most prevalent protozoan parasites of humans. We report the physical mapping of the genome of the Iowa isolate, sequencing and analysis of chromosome 6, and ∼0.9 Mbp of sequence sampled from the remainder of the genome. To construct a robust physical map, we devised a novel and general strategy, enabling accurate placement of clones regardless of clone artefacts. Analysis reveals a compact genome, unusually rich in membrane proteins. As in Plasmodium falciparum, the mean size of the predicted proteins is larger than that in other sequenced eukaryotes. We find several predicted proteins of interest as potential therapeutic targets, including one exhibiting similarity to the chloroquine resistance protein of Plasmodium. Coding sequence analysis argues against the conventional phylogenetic position of Cryptosporidium and supports an earlier suggestion that this genus arose from an early branching within the Apicomplexa. In agreement with this, we find no significant synteny and surprisingly little protein similarity with Plasmodium. Finally, we find two unusual and abundant repeats throughout the genome. Among sequenced genomes, one motif is abundant only in C. parvum, whereas the other is shared with (but has previously gone unnoticed in) all known genomes of the Coccidia and Haemosporida. These motifs appear to be unique in their structure, distribution and sequences.

HAPPY mapping is an in vitro approach for defining the order and spacing of DNA markers directly on native genomic DNA. This cloning-free technique is based on analysing the segregation of markers amplified from high molecular weight genomic DNA which has been broken randomly and 'segregated' by limiting dilution into subhaploid samples. It is a uniquely versatile tool, allowing for the construction of genome maps with flexible ranges and resolutions. Moreover, it is applicable to plant genomes, for which many of the techniques pioneered in animal genomes are inapplicable or inappropriate. We report here its demonstration in a plant genome by reconstructing the physical map of a 1.9 Mbp region around the FCA locus of Arabidopsis thaliana. The resulting map, spanning around 10% of chromosome 4, is in excellent agreement with the DNA sequence and has a mean marker spacing of 16 kbp. We argue that HAPPY maps of any required resolution can be made immediately and with relatively little effort for most plant species and, furthermore, that such maps can greatly aid the construction of regional or genome-wide physical maps.

The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome. Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes. This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb). If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.

Cryptosporidium parvum is a protozoan of the phylum Apicomplexa, class Coccidia. It is an obligate intracellular parasite infecting the gut epithelia of a wide range of mammals, and is transmitted from host to host via thick-walled oocysts in the faeces. The first case of human cryptosporidiosis was reported as recently as 1976, and it is now recognized as a major cause of gastro-intestinal illness worldwide. In most cases, the body mounts a strong immune response and the parasite is eradicated from the gut after ∼seven days of diarrhea. Infections in the immunosuppressed (e.g., AIDS patients), however, can be persistent and eventually fatal (for review, see Fayer et al. 1997). Massive outbreaks have occurred when the drinking water for entire communities has been contaminated with oocysts (e.g., MacKenzie et al. 1994). The Cryptosporidium genome is small for a coccidian (Tilley and Upton 1997), consisting of eight chromosomes with a total size estimate of between 9.6 (Caccio et al. 1998) and 10.4 Mb (Blunt et al. 1997). The low copy number and genome-wide distribution of both 5S and cytoplasmic rDNA genes (four to five and five copies, respectively; Taghi-Kilani et al. 1994; Le Blancq et al. 1997) is similar to that of several other apicomplexans, such as Plasmodium. This differs markedly from the usual eukaryotic pattern of hundreds or thousands of tandem copies. The ∼60%–70% AT content of the genome (Jenkins and Petersen 1997; Piper et al. 1998) is much lower than that of most Plasmodium spp. (Weber 1988). C. parvum appears to contain neither mitochondria (although they have been observed in other Cryptosporidium spp.) nor the plastid commonly found in apicomplexan parasites (Fayer et al. 1997; Kohler et al. 1997). Around 15–20 complete genes have been sequenced (e.g., Gooze et al. 1991; Nelson et al. 1991; Kim et al. 1992; Lally et al. 1992; Jenkins et al. 1993; Ranucci et al. 1993; Taghi-Kilani et al. 1994; Khramstov et al. 1995, 1996; Steele et al. 1995), and many others have been identified during the UCSF Cryptosporidium EST project (http://www.embl-ebi.ac.uk/parasites/news.html). Like most apicomplexans, Cryptosporidium appears to have few introns—currently only one has been reported (Caccio et al. 1997). The combination of its medical importance and small, unusual genome make C. parvum an ideal candidate for genomic studies, but these have been hampered by the absence of a detailed map and large insert clone library. The second of these problems was addressed recently with the release of the C. parvum PAC library (Piper et al. 1998). It has only recently become possible, however, to distinguish all eight Cryptosporidium chromosomes electrophoretically, and mapping to date has been limited to the placing of 20 markers on specific chromosomes (Caccio et al. 1998). This paper describes the construction of a physical genome map of C. parvum using HAPPY mapping (Dear and Cook 1993; Dear 1997; Dear et al. 1998), an in vitro technique that involves breaking intact genomic DNA at random, segregating the fragments into aliquots by limiting dilution and measuring the frequency of cosegregation of markers among the aliquots (Fig. ​(Fig.1).1). Closely linked markers are rarely separated by an intervening break and therefore tend to cosegregate. In this way, it is analogous both to traditional linkage mapping, which measures the frequency of recombination between markers during meiosis, and to radiation hybrid mapping (Cox et al. 1990). HAPPY mapping, however, does not suffer from the inaccuracies inherent in biological methods (recombination hotspots or hybrid instability), and the AT content of the genome should be largely immaterial. Constructing and screening a HAPPY mapping panel is relatively simple, and only small quantities of DNA are needed. All of the above suggest that HAPPY mapping is ideal for the study of otherwise intractable parasite genomes. Genomic studies of Cryptosporidium should be greatly aided by the use of this map in conjunction with the PAC library. Accession numbers and primer sequences for all markers are available at http://www.mrc-lmb.cam.ac.uk/happy/CRYPTO/crypto-genome.html. In addition, a limited number of aliquots of the mapping panel can be made available to researchers wishing to map their own markers. Figure 1 How HAPPY mapping works. Intact genomic DNA is broken by irradiation (I) to give a pool of random fragments, which are size selected by PFGE (II). A mapping panel of 96 aliquots is taken (III) from this pool. Each aliquot contains ∼1 haploid genome’s ...

Cryptosporidium par6um is an obligate-intracellular parasite of the gut mucosa. It infects many mammals, and can be considered an emerging pathogen in man, the first case of human cryptosporidiosis being reported as recently as 1976 [1]. Little is known about the molecular genetics of Cryptosporidium, partly due to our inability to continuously culture it in vitro. Oocysts for study are usually obtained by extraction from the faeces of infected calves or lambs [1]. Karyotype analysis suggests that the Cryptosporidium genome consists of eight chromosomes of :1–2 Mb [2,3], giving a genome size of 10.4 Mb [2]. Several partial and complete gene sequences have been reported [4–15], and an expressed sequence tag (EST) sequencing project is currently in progress (http:// www.embl-ebi.ac.uk/parasites/news.html). Several small insert Cryptosporidium libraries, both genomic [6,9] and cDNA [4,5,12–15], have been reported. However, no large-insert libraries have hitherto been available, impeding mapping and sequencing efforts. The vector chosen for constructing this library was the P1 artificial chromosome (PAC) vector pCYPAC2 [16], which differs by only a single NotI restriction site from pCYPAC1 [17]. PAC vectors are based on the P1 vector system [18,19], but are introduced into the host cell by electroporation rather than viral transformation. A PAC library can be manipulated exactly as one would a P1 library and has the same desirable features, e.g. clonal stability over many generations and Abbre6iations: EST, expressed sequence tag; PAC, P1 artificial chromosome; PFGE, pulsed field gel electrophoresis; STS, sequence tagged site. * Corresponding author. Tel.: +44 1223 402190; fax: +44 1223 412178; e-mail: phd@mrc-lmb.cam.ac.uk 1 Note: Nucleotide sequence data reported in this paper are available in the EMBL, DDJB and GenBankTM databases under the accession numbers G35127–G35275 and G35338– G35349.

A translocation involving chromosomes 12 and 14 [t(12;14)(q15;24.1)] is commonly seen in benign smooth muscle tumor as uterine leiomyoma (UL). A contig of P1-derived artificial chromosome and bacterial artificial chromosome clones on chromosome 14, encompassing a t(12;14) breakpoint cluster region (BCR) in UL, was generated principally using the recently developed HAPPY map of chromosome 14 as a framework (P. H. Dear et al., 1998, Genomics 48: 232-241). Three UL t(12;14) breakpoints have been localized within this contig, showing that a BCR of at least 400 kb exists on chromosome 14. Other studies of tumors with t(12;14) rearrangements similarly show breakpoints within a 475-kb multiple aberration region on chromosome 12. Thus t(12;14) is an example of a translocation in which the breakpoints are located within a BCR on both chromosome 12 and chromosome 14, justifying the identification of expressed sequences that are altered in these BCR regions. A total of four expressed sequences were identified in the BCR on chromosome 14. Two of these were novel cDNAs (D14S1460E and D14S1461E). The chromosome 14 cDNAs were expressed in multiple adult tissues. The identification of a large breakpoint cluster region on chromosome 14 suggests that translocations in this region mediate their effects at a distance and also that elements that predispose this region to recurrent chromosomal translocation may be widely distributed.

A new member of the NCAM family mapping to 3p12 has been isolated and predicted to be arranged in five immunoglobulin-like domains and three fibronectin-like domains which are particularly homologous to L1. There is a transmembrane domain and a long cytoplasmic region with no detectable homology to other sequences. Although less closely related to DCC, another family member, both share a loop of positively charged amino acids within the first immunoglobulin domain, unique to these two members of this very large gene family. Preliminary studies of expression in mouse embryos support an inferred role in neural development, but the observation of widespread gene expression in adult human tissues indicates that this protein has additional functions to those performed in neural cells.

We have mapped 1001 novel sequence-tagged sites on human chromosome 14. The mean spacing between markers is approximately 90 kb, most markers are mapped with a resolution of better than 100 kb, and physical distances are determined. The map was produced using HAPPY mapping, a simple and widely applicable in vitro approach that is analogous to linkage or to radiation hybrid mapping, but that circumvents many of the difficulties and potential artifacts associated with these methods. We show also that the map serves as a robust scaffold for building physical maps using large-insert clones.

Rationale: Amplification of distal 3q is the most common genomic aberration in squamous lung cancer (SQC). SQC develops in a multistage progression from normal bronchial epithelium through dysplasia to invasive disease. Identifying the key driver events in the early pathogenesis of SQC will facilitate the search for predictive molecular biomarkers and the identification of novel molecular targets for chemoprevention and therapeutic strategies. For technical reasons, previous attempts to analyze 3q amplification in preinvasive lesions have focused on small numbers of predetermined candidate loci rather than an unbiased survey of copy-number variation. Objectives: To perform a detailed analysis of the 3q amplicon in bronchial dysplasia of different histological grades. Methods: We use molecular copy-number counting (MCC) to analyze the structure of chromosome 3 in 19 preinvasive bronchial biopsy specimens from 15 patients and sequential biopsy specimens from 3 individuals. Measurements and Main Results: We demonstrate that no low-grade lesions, but all high-grade lesions, have 3q amplification. None of seven low-grade lesions progressed clinically, whereas 8 of 10 patients with high-grade disease progressed to cancer. We identify a minimum commonly amplified region on chromosome 3 consisting of 17 genes, including 2 known oncogenes, SOX2 and PIK3CA. We confirm that both genes are amplified in all high-grade dysplastic lesions tested. We further demonstrate, in three individuals, that the clinical progression of high-grade preinvasive disease is associated with incremental amplification of SOX2, suggesting this promotes malignant progression. Conclusions: These findings demonstrate progressive 3q amplification in the evolution of preinvasive SQC and implicate SOX2 as a key target of this dynamic process.

An automated gridding procedure for the inoculation of yeast and bacterial clones in high-density arrays has been developed. A 96-pin inoculating tool compatible with the standard microtiter plate format and an eight-position tablet have been designed to fit the Biomek 1000 programmable robotic workstation (Beckman Instruments). The system is used to inoculate six copies of 80 x 120-mm filters representing a total of approximately 20,000 individual clones in approximately 3 h. High-density arrays of yeast artificial chromosome (YAC) and cosmid clones have been used for rapid large-scale hybridization screens of ordered libraries. In addition, an improved PCR library screening strategy has been developed using strips cut from the high-density arrays to prepare row and column DNA pools for PCR analysis. This strategy eliminates the final hybridization step and allows identification of a single clone by PCR in 2 days. The development of automated gridding technology will have a significant impact on the establishment of fully versatile screening of ordered library resources for genomic studies.

The rate limiting step in a large-scale sequencing project is the generation of single-stranded DNA templates. We describe a fast, semiautomated procedure, using 96-well microtitre plates, in which 192 templates can be readily prepared in 1 day. The technique can be carried out manually or can be semiautomated using a robot pipetting device. We also provide evidence for the reliability and applicability of this method to a large-scale sequencing project.

Eimeria tenella is an intracellular protozoan parasite that infects the intestinal tracts of domestic fowl and causes coccidiosis, a serious and sometimes lethal enteritis. Eimeria falls in the same phylum (Apicomplexa) as several human and animal parasites such as Cryptosporidium, Toxoplasma, and the malaria parasite, Plasmodium. Here we report the sequencing and analysis of the first chromosome of E. tenella, a chromosome believed to carry loci associated with drug resistance and known to differ between virulent and attenuated strains of the parasite. The chromosome—which appears to be representative of the genome—is gene-dense and rich in simple-sequence repeats, many of which appear to give rise to repetitive amino acid tracts in the predicted proteins. Most striking is the segmentation of the chromosome into repeat-rich regions peppered with transposon-like elements and telomere-like repeats, alternating with repeat-free regions. Predicted genes differ in character between the two types of segment, and the repeat-rich regions appear to be associated with strain-to-strain variation.

Many methods exist for genotyping--revealing which alleles an individual carries at different genetic loci. A harder problem is haplotyping--determining which alleles lie on each of the two homologous chromosomes in a diploid individual. Conventional approaches to haplotyping require the use of several generations to reconstruct haplotypes within a pedigree, or use statistical methods to estimate the prevalence of different haplotypes in a population. Several molecular haplotyping methods have been proposed, but have been limited to small numbers of loci, usually over short distances. Here we demonstrate a method which allows rapid molecular haplotyping of many loci over long distances. The method requires no more genotypings than pedigree methods, but requires no family material. It relies on a procedure to identify and genotype single DNA molecules, and reconstruction of long haplotypes by a 'tiling' approach. We demonstrate this by resolving haplotypes in two regions of the human genome, harbouring 20 and 105 single-nucleotide polymorphisms, respectively. The method can be extended to reconstruct haplotypes of arbitrary complexity and length, and can make use of a variety of genotyping platforms. We also argue that this method is applicable in situations which are intractable to conventional approaches.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum , type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma , the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.

We have made a high-resolution HAPPY map of chromosome 6 of Dictyostelium discoideum consisting of 300 sequence-tagged sites with an average spacing of 14 kb along the approximately 4-Mb chromosome. The majority of the marker sequences were derived from randomly chosen clones from four different chromosome 6-enriched plasmid libraries or from subclones of YACs previously mapped to chromosome 6. The map appears to span the entire chromosome, although marker density is greater in some regions than in others and is lowest within the telomeric region. Our map largely supports previous gene-based maps of this chromosome but reveals a number of errors in the physical map. In addition, we find that a high proportion of the plasmid sequences derived from gel-enriched chromosome 6 (and that form the basis of a chromosome-specific sequencing project) originates from other chromosomes.

We have cloned the human full-length cDNA SEL1L, which is highly similar to the C. elegans sel-1 gene, an important negative regulator of the "notch" pathway which acts as a key regulator of the cellular proliferation and specification processes in both vertebrates and invertebrates. The SEL1L gene maps to 14q24.3–31 and here we report its fine localization by HAPPY mapping, which determines its molecular distance to microsatellite markers isolated in the region. We have found two new polymorphic (CA)n microsatellites located in the gene, and have identified the exon-intron boundaries. The gene is composed of 21 exons spanning 70 kb of genomic DNA. Human SEL1L protein exhibits a high degree of similarity compared to the mouse and nematode homologs.

We have localized the gene encoding human RNase k6 to within ∼120 kb on the long (q) arm of chromosome 14 by HAPPY mapping. With this information, the relative positions of the six human RNase A ribonucleases that have been mapped to this locus can be inferred. To further our understanding of the individual lineages comprising the RNase A superfamily, we have isolated and characterized 10 novel genes orthologous to that encoding human RNase k6 from Great Ape, Old World, and New World monkey genomes. Each gene encodes a complete ORF with no less than 86% amino acid sequence identity to human RNase k6 with the eight cysteines and catalytic histidines (H15 and H123) and lysine (K38) typically observed among members of the RNase A superfamily. Interesting trends include an unusually low number of synonymous substitutions (Ks) observed among the New World monkey RNase k6 genes. When considering nonsilent mutations, RNase k6 is a relatively stable lineage, with a nonsynonymous substitution rate of 0.40 × 10−9 nonsynonymous substitutions/nonsynonymous site/year (ns/ns/yr). These results stand in contrast to those determined for the primate orthologs of the two closely related ribonucleases, the eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), which have incorporated nonsilent mutations at very rapid rates (1.9 × 10−9and 2.0 × 10−9 ns/ns/yr, respectively). The uneventful trends observed for RNase k6 serve to spotlight the unique nature of EDN and ECP and the unusual evolutionary constraints to which these two ribonuclease genes must be responding.[The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF037081–AF037090.]

Publisher Summary This chapter discusses various preparation and fluorescent sequencing of M13 clones that is by the microtiter methods. Fluorescent sequencing is found to be the fastest and most efficient way to generate sequence data, when combined with microtiter protocols for template preparation and sequencing reactions. DNA fragments up to 134 kb in size; 21 have been sequenced in a single shotgun, in which the DNA fragment is subcloned in M13 and assembled as one contiguous sequence. The development of a semi-automated microtiter protocol for template preparation overcomes a rate-limiting step in large sequencing projects. The current limitation in the procedure described, using the Applied Biosystems 373A, is gel throughput. However, this method offers significant advantages. Particularly, the manual digitizing of autoradiographic images is no longer necessary, and it consistently obtains greater than 400 nucleotides of accurate sequence per clone. The combination of this base-calling capability with high-throughput microtiter methods for template preparation and sequencing reactions is an effective implementation of the shotgun strategy.

The DNA sequence of Raji DNA spanning the deletion found in B95-8 cells has been determined. Three open reading frames and a region of homology with the BamHI-H fragment are found within the deletion. The deletion contains a region of 102-bp repeats which is transcribed into an mRNA. The Raji sequence reported here varies slightly from a smaller M-ABA sequence reported previously. This paper completes the sequence of all parts of the wild-type Epstein-Barr virus genome.

Large-scale sequence analysis of the AD169 strain of human cytomegalovirus (HCMV) began in this laboratory in 1984 when very little was known about the sequence or location of genetic information in the viral genome. At that time sequence analysis was confined to the major immediate-early gene (Stenberg et al. 1984), a region of the Colburn strain that contained CA tracts (Jeang and Hayward 1983), the L-S junction region (Tamashiro et al. 1984), and what has been termed the transforming region (Kouzarides et al. 1983). This chapter is being written in March 1989 when the sequence is complete except for some remaining polishing of certain areas which is still going on (manuscript in preparation). As far as we know there are no major discrepancies in the data which might lead to the sequence changing although of course this cannot be ruled out. We present a preliminary analysis of the HCMV genome and limit ourselves mainly to the potential protein-coding content of over 200 reading frames.

A human cytomegalovirus (HCMV) glycoprotein gene with homology to glycoprotein B (gB) of herpes simplex virus and Epstein-Barr virus and gpII of varicella zoster virus has been identified by nucleotide sequencing. The gene has been expressed in recombinant vaccinia virus and the gene product recognized by monoclonal antibodies and human immune sera. Rabbits immunized with the recombinant vaccinia virus produced antibodies that immunoprecipitate gB from HCMV-infected cells and neutralize HCMV infectivity in vitro. These data demonstrate a role for this protein in future HCMV vaccines.

The 12-kbp short unique region of the B95-8 Epstein-Barr virus (EBV) genome has been sequenced and analysed for latent and lytic cycle transcripts. Two latent and three late mRNAs have been detected, the largest of the late transcripts potentially encoding a 143-kDa protein. The region containing oriP, the putative origin of replication of the genome as a plasmid in latently infected B lymphocytes, is shown to contain 21 direct repeats of a 30-bp A+T-rich sequence and a related large inverted repeat.

A small proportion (0.1-0.5%) of the total DNA content of native Chinese hamster metaphase chromosomes is protected from nucleolytic degradation following the removal of histones by extraction with either 0.2 N HCl or 2 M NaCl, and remains attached to the nonhistone protein core. Acid extraction followed by DNase I digestion leads to small fragments of 10-30 bases. Salt extraction followed by micrococcal nuclease digestion gives approx. 140 b.p. fragments which are undistinguishable in size from nucleosome core DNA fragments. Furthermore, DNase I treatment of salt extracted chromosomes gives DNA fragments containing single strands which are multiples of 10 bases in length, again characteristic of the nucleosome structure. Reassociation kinetics using the 32P-labelled 140 b.p. fragments as probes suggests they are enriched for rapidly reassociating sequences.

The complete (172,282 base pairs) nucleotide sequence of the B95-8 strain of Epstein-Barr virus has been established using the dideoxynucleotide/M13 sequencing procedure. Many RNA polymerase II promoters have been mapped and the mRNAs from these promoters have been assigned to the latent or early/late productive virus cycles. Likely protein-coding regions have been identified and three of these have been shown to encode a ribonucleotide reductase, a DNA polymerase and two surface glycoproteins.

The structural gene for yeast DNA topoisomerase I (TOP1) has been cloned from two yeast genomic plasmid banks. Integration of a plasmid carrying the gene into the chromosome and subsequent genetic mapping shows that TOP1 is identical to the gene previously called MAK1. Seven top1 (mak1) mutants including gene disruptions are viable, demonstrating that DNA topoisomerase I is not essential for viability in yeast. A 3787-base-pair DNA fragment including the gene has been sequenced. The protein predicted from the DNA sequence has 769 amino acids and a molecular weight of 90,020.

Comparison of the human mitochrondrial DNA sequence of the cytochrome oxidase subunit II gene and the sequence of the corresponding beef heart protein shows that UGA is used as a tryptophan codon and not as a termination codon and suggests that AU A may be a methionine and not an isoleucine codon. The cytochrome oxidase II gene is contiguous at its 5′ end with a tRNAAsp gene and there are only 25 bases at its 3′ end before a tRNALys gene. These tRNAs are different from all other known tRNA sequences.

DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. The authors present here a 5280 base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, they were able to analyze the extent of homology between the DNA polymerases of three distantly related herpes viruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpes viruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type I and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barrmore » virus.« less

The H-DNA repeat unit of Herpesvirus saimiri strain 11 was cloned in plasmid vector pAGO, and the nucleotide sequence was determined by the dideoxy chain termination method. One unit of repetitive DNA has 1,444 base pairs with 70.8% G+C content. The structural features of repeat DNA sequences at the termini of intact virion M-DNA (160 kilobases) and orientation of reiterated DNA were analyzed by radioactive end labeling of M-DNA, followed by cleavage of the end fragments with restriction endonucleases. The termini appeared to be blunt ended with a 5'-phosphate group, probably generated during encapsidation by cleavage in the immediate vicinity of the single ApaI recognition site in the H-DNA repeat unit. The sequence did not reveal sizeable open reading frames, the longest hypothetical peptide from H-DNA being 85 amino acids. There was no evidence for an mRNA promoter or terminator element, and H-DNA-specific transcription could not be found in productively infected cells.

The 20,349-bp sequence of the human cytomegalovirus (HCMV) HindIII F fragment has revealed eight open reading frames with homology to herpes simplex virus (HSV) and/or Epstein-Barr virus (EBV). With respect to EBV, these homologous genes can be divided into two blocks: one block contains three genes, including the DNA polymerase and glycoprotein B, and the other block contains five genes of unknown function. Although the relative organisation of genes within each block is identical in HCMV and EBV, the relative position of each block within the two genomes differs: in HCMV the two blocks are present directly adjacent to each other, whereas in EBV they are found 92 kb apart. This suggests that a genetic rearrangement has occurred in this region. Transcription analysis of the glycoprotein B gene is presented and the evolutionary relationship between the genomes of HCMV, EBV, and HSV is discussed.

The yeast HO gene, which encodes an endonuclease involved in initiating mating type interconversion, is expressed in mother cells but not in daughters. It has been demonstrated that the SWI5 gene, which is an activator of HO expression, plays a critical role in this differential mother/daughter expression of HO. In this paper we describe the cloning and sequencing of the SWI5 gene. The predicted amino acid sequence derived from the cloned SWI5 gene shows homology with the repeated DNA-binding domains ('zinc fingers') of Xenopus transcription factor TFIIIA. A region of the HO promoter involved in the SWI5-dependent transcriptional activation of HO was identified by deletion analysis of the HO promoter in the chromosome, and by testing the ability of HO DNA fragments to activate transcription in the context of a heterologous promoter. The SWI5 gene product was overproduced in yeast from the GAL1-10 promoter, since the SWI5 protein is made at very low levels in wild-type strains, and protein extracts were used to demonstrate that the SWI5 protein binds in vitro to a segment of the HO promoter required for transcriptional activation in vivo.

The DNA sequence of the EcoRI Dhet and part of the BamHI E fragments of Raji EBV has been determined. Precise locations of two deletions in Raji DNA have been identified and their consequences for gene structure evaluated. The deletion in Raji of reading frames BALF1, BARF1, and BZLF2 and truncation of BALF2 and BERF5 probably account for the replication defect in this strain. The degree of sequence variation between B95-8 and Raji has been examined and shows considerable variation between genes. The latent membrane protein gene is exceptionally polymorphic and the initiator methionine for the late productive cycle protein overlapping the latent membrane protein is absent in Raji.

The 17,180 base-pair Eco-RI-C fragment of Epstein-Barr virus has been sequenced in its entirety. This same fragment has also been analyzed for RNA polymerase II promoters, which are active in a soluble in vitro assay. These data are compared to the availability of predicted open reading frames and potential nucleotide signals associated with transcription. In addition, the DNA sequence of a number of previously undetected repeated DNA sequences from this and several nearby regions of the viral genome are reported.

Analysis of an almost complete mammalian mitochondrial DNA sequence has identified 23 possible tRNA genes and we speculate here that these are sufficient to translate all the codons of the mitochondrial genetic code. This number is much smaller than the minimum of 31 required by the wobble hypothesis. For each of the eight genetic code boxes with four codons for one amino acid we find a single specific tRNA gene with T in the first (wobble) position of the anticodon. We suggest that these tRNAs with U in the wobble position can recognize all four codons in these genetic code boxes either by a "two out of three" base interaction or by U.N wobble.

The sequence of a region of the HCMV genome transcribed during the immediate early (IE) transcriptional phase has been determined. Transcription analysis of this region at the junction between fragments HindIII Z and J has identified three moderately abundant mRNAs of 3.4, 1.7, and 1.65 kb. The 3.4-kb RNA is expressed only under IE conditions of infection. It is composed of four exons and its predicted translation product has features characteristic of a membrane-bound glycoprotein. The 1.7-kb RNA is transcribed at both IE and late times postinfection. It is expressed from the same promoter as the 3.4-kb RNA and does not appear to be spliced. The 1.65-kb RNA is present at the IE phase, but is more abundantly transcribed at late times. It is composed of two exons and is 3′ coterminal to the 3.4-kb RNA. Within the predicted translation product of the 1.65-kb RNA there are three regions which show homology to a family of related open reading frames found within the short unique region of HCMV.

The human mitochondrial (mt) genome consists of a closed circular duplex DNA approximately 10 x 106 daltons and has been the most intensely studied animal mt genetic system. The positions of the origin of replication of H strand synthesis (Crews et al. 1979), the 12S and 16S ribosomal RNA genes (Robberson et al. 1972) and 19 tRNA genes (Angerer et al. 1976) have been located on the genetic map shown in Figure 1. A number of discrete products of mitochondrial protein synthesis have been demonstrated and three of them identified as subunits 1, 2 and 3 of the cytochrome oxidase complex (Hare et al. 1980). In comparison with other mito-systems, genes for up to four subunits of the ATPase complex, one of the cytochrome bc1 complex and possibly for a ribosomal protein would be expected to be present (see review by Borst 1977). Both strands are thought to be completely transcribed symmetrically from a point near the origin of the H strand synthesis (Aloni and Attardi 1971; Murphy et al. 1975). These transcripts are then processed to give the rRNAs, the tRNAs and a number of polyadenylated but not capped mRNAs (Attardi et al. 1979). Both the L and H strands have been shown to be coding with the L strand containing the sense sequence of the rRNA genes, most of the tRNA genes and most of the stable polyadenylated mRNAs.

The complete sequence of the 16,569-base pair human mitochondrial genome is presented. The genes for the 12S and 16S rRNAs, 22 tRNAs, cytochrome c oxidase subunits I, II and III, ATPase subunit 6, cytochrome b and eight other predicted protein coding genes have been located. The sequence shows extreme economy in that the genes have none or only a few noncoding bases between them, and in many cases the termination codons are not coded in the DNA but are created post-transcriptionally by polyadenylation of the mRNAs.

The sequence of the 17,166-base-pair EcoRI C fragment of Epstein-Barr virus DNA, cell line B95-8, was determined. In vitro transcription was used to identify three RNA polymerase II promoters within this fragment of the virus. Cytoplasmic poly(A)+ RNAs starting at these points were demonstrated in B95-8 cells induced into virus production with 12-O-tetradecanoylphorbol 13-acetate. Uninduced B95-8 cells contained much less of these RNAs. The upstream sequences near the three promoters show striking homologies that may be involved in transcriptional control.