DNA Sequence Determination Research Articles

Molecular cloning, characterization and regulation of a peroxiredoxin gene from Schizosaccharomyces pombe

A gene encoding a putative peroxiredoxin (Prx) of the fission yeast Schizosaccharomyces pombe was characterized and its regulation was studied. The full length of the prx gene was introduced into the shuttle vector pRS316 after PCR amplification, resulting in the recombinant plasmid pPrx10. The determined DNA sequence carries 1,327 bp encoding a putative Prx with a molecular mass of 19,510 Da. Prx activity was significantly increased in the S. pombe cells harboring pPrx10. The accelerated growth was observed in the S. pombe/pPrx10 cells, implying the involvement of the cloned gene in the yeast growth. To study transcriptional regulation of the prx gene, the prx-lacZ fusion gene was constructed using the yeast-E. coli shuttle vector YEp367R, and named pPrxup10. The synthesis of beta-galactosidase from the fusion gene was enhanced under carbon source-limited conditions and nitrogen starvation. Under the same growth conditions, the prx mRNA levels of the wild-type yeast cells were increased. The prx mRNA level was markedly decreased in the Pap1-negative mutant, compared with that in the wild-type yeast, suggesting that the basal expression of the prx gene is mediated by a transcription factor, Pap1. The reactive oxygen species (ROS) level was diminished in the S. pombe/pPrx10 cells than in the control cells. The extra copies of the prx gene were able to resist elevation of ROS level under limited carbon source condition and menadione treatment. In brief, the S. pombe Prx is linked with the yeast growth and up-regulated by metabolic oxidative stress on a transcriptional level. The Prx protein is partly responsible for maintaining low ROS level under normal and stressful growth conditions in the fission yeast.

The Fluoride Cleavable 2-(Cyanoethoxy)Methyl (CEM) Group as Reversible 3′-O-Terminator for DNA Sequencing-by-Synthesis—Synthesis, Incorporation, and Cleavage

A new and promising sequencing technology called sequencing-by-synthesis (SBS) enables fast determination of DNA sequences. 2′-Deoxynucleotides containing the (2-cyanoethoxy)methyl (CEM) group at the 3′-O-position are potential reversible terminators for the SBS technology. Herein we describe the synthesis, the incorporation by several polymerases, and the cleavage of this 3′-O-blocking group using 3′-O-CEM-thymidinyl-5′-O-triphosphate 7 as an example.

Determination of a Minimal DNA Sequence of the Internal Transcribed Spacer Region for the in silico Identification of Botryosphaeria sp.

Determination of a Minimal DNA Sequence of the Internal Transcribed Spacer Region for the in silico Identification of Botryosphaeria sp.

Cloning, sequencing and molecular characterisation of a cryptic plasmid from a urease-positive thermophilic Campylobacter (UPTC) isolate

Cloning, sequencing and molecular characterisation of a cryptic plasmid, pUPTC237, from a urease-positive thermophilic Campylobacter (UPTC) isolate obtained from the natural environment in Northern Ireland is reported in this study. Based on the determined DNA sequence, the pUPTC237 DNA was identified as a circular molecule of 3828 bp with a G+C content of 29.5%. As with other plasmid DNAs from Gram-negative bacteria, pUPTC237 contained an A+T-rich region (A+T content: 95%), followed by multiple direct tandem repeat units of 22 bp, characteristic of a replication origin and iteron sequence. A possible open reading frame (ORF)-1 was located upstream of the A+T-rich region and the iteron sequence that encoded a 460 amino acid protein similar to the mobilisation (mob) protein and two putative promoter structure sequences at the -35 and -10 regions and a possible ribosome binding site occurred upstream of the start codon for the ORF-1. Moreover, three possible ORFs (a short ORF-2 encoding 26 amino acids, similar to repA; an ORF-3 encoding 341 amino acids, similar to repB; and an ORF-4 encoding 96 amino acids with unknown function) were also identified. There are also two putative promoter structures for these three ORFs at the -35 and -10 regions upstream of the possible ORF-2. A possible transcription termination region was identified downstream of ORF-4. Northern blot hybridisation analysis suggested that these four ORFs constitute an operon and generate a messenger RNA (mRNA) transcript.

Protein and DNA sequence determinants of thermophilic adaptation.

There have been considerable attempts in the past to relate phenotypic trait—habitat temperature of organisms—to their genotypes, most importantly compositions of their genomes and proteomes. However, despite accumulation of anecdotal evidence, an exact and conclusive relationship between the former and the latter has been elusive. We present an exhaustive study of the relationship between amino acid composition of proteomes, nucleotide composition of DNA, and optimal growth temperature (OGT) of prokaryotes. Based on 204 complete proteomes of archaea and bacteria spanning the temperature range from −10 °C to 110 °C, we performed an exhaustive enumeration of all possible sets of amino acids and found a set of amino acids whose total fraction in a proteome is correlated, to a remarkable extent, with the OGT. The universal set is Ile, Val, Tyr, Trp, Arg, Glu, Leu (IVYWREL), and the correlation coefficient is as high as 0.93. We also found that the G + C content in 204 complete genomes does not exhibit a significant correlation with OGT (R = −0.10). On the other hand, the fraction of A + G in coding DNA is correlated with temperature, to a considerable extent, due to codon patterns of IVYWREL amino acids. Further, we found strong and independent correlation between OGT and the frequency with which pairs of A and G nucleotides appear as nearest neighbors in genome sequences. This adaptation is achieved via codon bias. These findings present a direct link between principles of proteins structure and stability and evolutionary mechanisms of thermophylic adaptation. On the nucleotide level, the analysis provides an example of how nature utilizes codon bias for evolutionary adaptation to extreme conditions. Together these results provide a complete picture of how compositions of proteomes and genomes in prokaryotes adjust to the extreme conditions of the environment.

The determination of multiple microsatellite genotypes and DNA sequences from a single pollen grain

AbstractPollen plays important roles in the reproduction and gene flow of flowering plants, and its haploid DNA sequence provides useful information for studies of plant evolution and genealogy. We describe a new method for multiple microsatellite genotyping and DNA sequencing from a single pollen grain. The haploid DNA was extracted from a single pollen grain by using a simple DNA extraction method, and multiple microsatellite genotypes and DNA sequences of multiple chloroplast loci were determined. Using nine pairs of microsatellite primers, more than 90% of genotypes were successfully determined, and 71% and 100% of DNA sequences were determined at two chloroplast DNA loci, the trnL intron region and the trnL/trnF intergenic spacer region, respectively. This simple method of genetic analysis for a single pollen grain will facilitate detailed study of pollination, evolution and genealogy.

Clonal Expansion of MLL-ELL Rearrangement without Relapse of Leukemia after Treatment of MLL-ELL Positive AML; with a Rare Breakpoint in MLL Gene.

Introduction The MLL gene located on 11q13 is a frequent target of chromosomal translocation and induces acute leukemia with a poor prognosis. The ELL gene, one of partner genes with MLL translocation, is located on 19p13.1 and involved in about 5% of spontaneous acute leukemias and about 30% of secondary acute leukemias with MLL gene rearrangement. Here we present a case who had clonal expansion of MLL-ELL- positive cells in bone marrow (BM) with normal morphology after achieving complete remission (CR) and successfully underwent unrelated bone marrow transplantation (UR-BMT). We also analyzed the breakpoint of MLL-ELL by RT-PCR and sequencing and identified a novel type of breakpoint in MLL gene.Patient and methods A 32-years-old Japanese woman, who had no previous medical history, suffered from dyspnea and severe pancytopenia and referred to a hospital in Dec. 2001. BM aspiration showed hypercellular marrow with 89% of blasts which were positive for myeloperoxidase staining and expressed myeloid immunophenotypes (i.e. CD7, CD13, CD33, CD34 and HLA-DR), leading to the diagnosis of AML. BM cytogenetics revealed t(11;19)(q23;p13.1), along with trisomy 8. After one course of induction therapy, she achieved CR. BM cytogenetics showed normal chromosome (46, XX) and FISH analysis of MLL gene showed negative for MLL rearranged cells. Consolidation chemotherapies were carried out and BM aspirations showed CR throughout the entire course. However, repeated FISH analyses revealed the number of MLL rearrangement positive cells were gradually increasing without expanding of leukemic blasts. Since MLL-ELL positive leukemia shows a very poor prognosis, UR-BMT was planned through Japan Marrow Donor Program. She was re-admitted on April 2003. Before transplantation, her complete blood counts were normal and BM aspirate showed hypercellular marrow with 0.4% blasts with myeloid/erythroid ratio 4.5. There were no morphological abnormalities, however, cytogenetic study showed 100 % of cells were positive for t(11;19)(q23;p13.1) without trisomy 8. FISH analysis showed 90.1% of cells were positive for MLL rearrangement and negative for trisomy 8. She underwent UR-BMT successfully and is still alive in good health. We analyzed MLL-ELL gene rearrangement by RT-PCR and determined DNA sequence of MLL-ELL fusions.Results We obtained unexpected longer PCR products than that of previously reported and sequencing analysis revealed exon 12 of MLL gene fused to ELL gene, although it has been reported that exons 10 or 11 of MLL is fused in frame with ELL in most cases of MLL-ELL translocations.Discussion We identified new type of MLL gene breakpoint which contains exon 12 of MLL gene in MLL-ELL positive AML. This type of MLL fusion, which contains an intact PHD domain, may cause clonal expansion of MLL-ELL rearrangement positive cells with no obvious differentiation block. Now, we are analyzing biological differences between common type MLL-ELL and exon12 type MLL-ELL by using retroviral gene transfer system.

Cloning, characterization and regulation of a protein disulfide isomerase from the fission yeast Schizosaccharomyces pombe

To elucidate the physiological roles and regulation of a protein disulfide isomerase (PDI) from the fission yeast Schizosaccharomyces pombe, the full-length PDI gene was ligated into the shuttle vector pRS316, resulting in pPDI10. The determined DNA sequence carries 1,636 bp and encodes the putative 359 amino acid sequence of PDI with a molecular mass of 39,490 Da. In the amino acid sequence, the S. pombe PDI appears to be very homologous to A. thaliana PDI. The S. pombe cells harboring pPDI10 showed increased PDI activity and accelerated growth, suggesting that the cloned PDI gene is functioning and involved in the yeast growth. The 460 bp upstream region of the PDI gene was fused into promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate pYUPDI10. The synthesis of beta-galactosidase from the PDI-lacZ fusion gene was enhanced by oxidative stress, such as superoxide anion and hydrogen peroxide. It was also induced by some non-fermentable and fermentable carbon sources. Nitrogen starvation was able to enhance the synthesis of beta-galactosidase from the PDI-lacZ fusion gene. The enhancement by oxidative stress and fermentable carbon sources did not depend on the presence of Pap1. The PDI mRNA levels were increased in both Pap1-positive and Pap1-negative cells treated with glycerol. Taken together, the S. pombe PDI gene is involved in cellular growth and response to nutritional and oxidative stress.

Rapid cost-effective subtyping of meticillin-resistant Staphylococcus aureus by denaturing HPLC

The importance of meticillin-resistant Staphylococcus aureus (MRSA) in hospital-acquired infection is widely acknowledged. The UK government has stated that MRSA bloodstream infection rates will have to be halved by 2008. Such radical improvements will require advances on several fronts. Screening for MRSA in high-risk patients on arrival at hospital allows isolation of carriers and reduces transmission to staff and other patients. Concurrent subtyping of MRSA could also inform outbreak investigations and long-term epidemiological studies. The variability within the staphylococcal protein A, or spaA, gene-repeat region can be used as a marker of short- and long-term genetic variation. A novel application is described of denaturing HPLC (DHPLC) for rapid, inexpensive characterization of spaA gene amplification products, without the need for DNA sequence determination. The method allowed rapid and precise sizing of spaA gene-repeat regions from 99 S. aureus strains and was combined with heteroduplex analysis, using reference PCR products, to indicate the spa type of the test isolate. The method allowed subtyping of strains in less than 5 h from receipt of a primary isolation plate. When applied to an outbreak that occurred during this study, the authors were able to demonstrate relatedness of the isolates more than 5 days before results were received from a reference laboratory. If combined with direct amplification from swabs, DHPLC analysis of spaA gene variation could prove extremely valuable in outbreak investigation and MRSA surveillance.

Isolation and molecular characterization of the porcine transforming growth factor beta type I receptor ( TGFBR1) gene

The transforming growth factor beta (TGF beta) family is essential for normal growth and development of different organ systems. Here we describe the isolation and molecular characterization of the full-length cDNA and the determination of the genomic DNA sequence of the porcine TGFBR1 gene. The full-length TGFBR1 cDNA 1813 bp contains an open reading frame (ORF) of 1512 bp encoding a TGFBR1 protein of 503 amino acids with a calculated molecular weight (Mw) of 56.4 kDa. A BAC clone harboring the porcine TGFBR1 gene was isolated and sequenced. The results of genomic and cDNA sequences of the porcine TGFBR1 gene demonstrated that it spans a transcription unit of 62,182 bp consisting of nine exons ranging from 125 to 354 bp, and eight introns ranging from 1003 to 29,441 bp. A shorter porcine TGFBR1 isoform resulting from the alternative splicing of exon 7 in porcine TGFBR1 cDNA was detected. The shorter TGFBR1 isoform contained a 1140 bp ORF encoding 379 amino acids with a calculated Mw of 41.7 kDa. The core promoter of porcine TGFBR1 gene lacks a TATA box but contains GC boxes and CAAT boxes. Multiple transcription initiation and termination sites were identified in untranslated regions (UTR) resulting in the size of 5′-UTR varying from 15 to 62 bp, and the length of 3′-UTR varying from 169 to 228 bp. Quantitative real time PCR results showed that the TGFBR1 transcript was ubiquitously expressed in all tissues examined (i.e. fat, adrenal, brain, spinal cord, muscle, mandibular lymph node, thymus, bone marrow, uterus, spleen, testis, kidney, liver, and ovary). A total of eighty-five gene polymorphisms (77 SNPs and 8 indels) were detected in the porcine TGFBR1 gene by utilizing a panel of DNA from eight diversified pig breeds (Yorkshire, Chinese Meishan, Berkshire, Duroc, Hampshire, Landrace, Large White and Pietrain). The minor allele frequencies of these nucleotide variations varied from 0.13 to 0.5 with an average of 0.26. In addition, seventeen microsatellites were identified throughout the genomic sequence of the porcine TGFBR1 gene.

Identification and molecular detection of a deletion mutation responsible for a truncated cadherin of Helicoverpa armigera

Cadherins are a class of receptor proteins for Bacillus thuringiensis Cry1A toxins. Disruption of a cadherin gene ( Ha_BtR) is associated with Cry1Ac resistance in the cotton bollworm Helicoverpa armigera [Xu, X., Yu, L., Wu, Y., 2005. Disruption of a cadherin gene associated with resistance to Cry1Ac δ-endotoxin of B. thuringiensis in H. armigera. Appl. Environ. Microbiol. 71, 948–954]. Determination of the genomic DNA sequences of Ha_BtR gene showed that the wild type Ha_BtR coding sequence is comprised of 34 exons. A deletion between Exon 8 and Exon 25 was found to be responsible for a truncated cadherin in the Cry1Ac-resistant GYBT strain of H. armigera. The mutant allele of Ha_BtR ( r1) has two possible transcription variants, both of which produce the same truncated protein. A DNA-based detection method specific to the r1 allele was developed. This study will facilitate the monitoring of cadherin mutant frequency in field populations of H. armigera.

Nrf2 Negatively Regulates Osteoblast Differentiation via Interfering with Runx2-dependent Transcriptional Activation

Nrf2 (nuclear factor E2 p45-related factor 2) is believed to be a transcription factor essential for the regulation of many detoxifying and antioxidative genes in different tissues. In the present study, we investigated the role of Nrf2 in the regulation of osteoblastic differentiation. nrf2 mRNA expression was significantly up-regulated in femur isolated from ovariectomized mice, whereas in situ hybridization analysis revealed that up-regulation of nrf2 mRNA was mainly found in osteoblasts attached on cancellous bone in femur of ovariectomized mice. Expression of Nrf2 protein was also seen in osteoblasts in neonatal mouse tibia and calvaria. In osteoblastic MC3T3-E1 cells stably transfected with nrf2 expression vector, significant inhibition was seen in the maturation-dependent increase in alkaline phosphatase activity as well as the mineralized matrix formation. Stable overexpression of nrf2 significantly impaired Runx2 (runt-related transcription factor 2)-dependent stimulation of osteocalcin promoter activity and recruitment of Runx2 on osteocalcin promoter without affecting the expression of runx2 mRNA. Coimmunoprecipitation and mammalian two-hybrid assay revealed a physical interaction between Runx2 and Nrf2, whereas cellular distribution of endogenous Runx2 was not apparently changed by nrf2 overexpression in MC3T3-E1 cells. Alternatively, Nrf2 bound to antioxidant-responsive element-like-2 sequence of osteocalcin promoter. The inhibition by nrf2 on runx2-dependent osteocalcin promoter activity was partially prevented by the introduction of reporter of deletion mutant for ARE-like-2 sequence of osteocalcin promoter. These data suggest that Nrf2 may negatively regulate cellular differentiation through inhibition of the Runx2-dependent transcriptional activity in osteoblasts.

Alu-mediated 100-kb deletion in the primate genome: The loss of the agouti signaling protein gene in the lesser apes

Agouti signaling protein (ASIP) is an endogenous antagonist of melanocortin receptors that controls a wide range of physiological functions. Its central role in regulation of the melanocortin system implied that ASIP has been relevant to the evolution of various physiological traits in primates. In this study, we have tried to determine DNA sequences of the ASIP gene (ASIP) of various simian species to find molecular evolutionary aspects of ASIP. Unexpectedly, we found that the whole coding region of ASIP was missing only from the gibbon genome; gibbons constitute a large group of hominoid species in Southeast Asia. Our analyses revealed that unequal homologous recombination mediated by two AluSx elements erased a approximately 100-kb region including ASIP from the gibbon genome. The data provide new evidence for the significant roles of Alu elements in the dynamic evolution of the primate genome.

The ever-expanding neuropeptide gene families in the nematode Caenorhabditis elegans

Neuropeptides act as chemical signals in the nervous system to modulate behaviour. With the ongoing EST projects and DNA sequence determination of different genomes, the identification of neuropeptide genes has been made easier. Despite the relatively 'simple' repertoire of behaviours in the nematode Caenorhabditis elegans, this worm contains a surprisingly large and diverse set of neuropeptide genes. At least 109 genes encoding over 250 potential neuropeptides have been identified in C. elegans; all genes are likely to be expressed and many, if not all, of the predicted peptides are produced. The predicted peptides include: 38 insulin-like peptides, several of which are involved in development and reproductive growth, and over 70 FMRFamide-related peptides, some of which are involved in locomotion, reproduction, and social behaviour. Many of the C. elegans peptides are identical or highly similar to those isolated or predicted in parasitic nematodes, such as Ascaris suum, Haemonchus contortus, Ancylostoma caninum, Heterodera glycines and Meloidogyne arenaria, suggesting that the function of these peptides is similar across species. The challenge for the future is to determine the function of all the genes and individual peptides and to identify the receptors through which the peptides signal.

Common and Variable Contributions of Fis Residues to High-Affinity Binding at Different DNA Sequences

Fis is a nucleoid-associated protein that interacts with poorly related DNA sequences with a high degree of specificity. A difference of more than 3 orders of magnitude in apparent Kd values was observed between specific (Kd, approximately 1 to 4 nM) and nonspecific (Kd, approximately 4 microM) DNA binding. To examine the contributions of Fis residues to the high-affinity binding at different DNA sequences, 13 alanine substitutions were generated in or near the Fis helix-turn-helix DNA binding motif, and the resulting proteins were purified. In vitro binding assays at three different Fis sites (fis P II, hin distal, and lambda attR) revealed that R85, T87, R89, K90, and K91 played major roles in high-affinity DNA binding and that R85, T87, and K90 were consistently vital for binding to all three sites. Other residues made variable contributions to binding, depending on the binding site. N84 was required only for binding to the lambda attR Fis site, and the role of R89 was dramatically altered by the lambda attR DNA flanking sequence. The effects of Fis mutations on fis P II or hin distal site binding in vitro generally correlated with their abilities to mediate fis P repression or DNA inversion in vivo, demonstrating that the in vitro DNA-binding effects are relevant in vivo. The results suggest that while Fis is able to recognize a minimal common set of DNA sequence determinants at different binding sites, it is also equipped with a number of residues that contribute to the binding strength, some of which play variable roles.

Direct prediction of HP sequences of compact polymer chains from elastic force

In this paper, a new method is proposed to predict HP sequences of compact polymer chains from elastic force based on the PERM (pruned-enriched-Rosenbluth method) simulation. Two different HP sequences are selected here, and we let them pass through a nanopore after acting of elastic force, which only allow single but not double strands to pass. Some thermodynamics properties of compact polymer chains are investigated during the translocation process, such as average Helmholtz free energy 〈 A〉, average energy per bond 〈 U〉, and average contact energy per bond 〈 U c〉. We find that the curves of them change non-monotonously with different steps, which inversely can be used to distinguish H and P accurately. The most important parameter of them is elastic force f because it can be measured directly using single-molecule force spectroscopy (SMFS) in experiment. Through recording and comparing force-extension curves, we can determine the HP sequences accurately. This method is also applied to determine DNA sequence directly, and to study the contact interactions in proteins and the protein folding.

Characterization of two small cryptic plasmids from Pseudomonas sp. strain S-47

Two small cryptic plasmids, p47L and p47S, identified in Pseudomonas sp. S-47 were characterized by determination of DNA sequences and physical and functional maps. They are 3084 and 1782 bp in length, respectively, with GC contents of 63.55 and 65.21%. The detection of single-strand DNAs of both plasmids indicates that they replicate by a rolling-circle mechanism. The deduced polypeptide encoded by the rep gene of p47L is homologous with Rep proteins of plasmids belonging to the pIJ101/pJV1 family, which are known to replicate by the rolling-circle mechanism. Despite containing a homologous signature with Rep proteins of rolling-circle replicating (RCR) plasmids in the pT181 family, the Rep of p47S lacks significant homology with Rep proteins of this family and is missing a region similar to the family’s replication origin ( dso). Based on the rep sequence comparisons, p47L falls into a previously defined plasmid family whereas p47S defines a new family of RCR plasmid.

GENETIC COMPLEMENTATION TO IDENTIFY DNA ELEMENTS THAT INFLUENCE COMPLEMENT RESISTANCE IN LEISHMANIA CHAGASI

Past studies showed that Leishmania spp. promastigotes exhibit differential sensitivity to complement mediated lysis (CML) during development in vitro and in vivo. Leishmania chagasi promastigotes in cultures during logarithmic and stationary growth phases are CML-sensitive or CML-resistant when exposed to human serum, respectively, but only in cultures recently initiated with parasites from infected animals; serially passaged cultures become constitutively CML-sensitive regardless of growth phase. Building on these observations, a genetic screen was conducted to identify novel complement resistance factors of L. chagasi. A cosmid library containing genomic DNA was transfected into a promastigote line previously subjected to >50 serial passages. Selection with human serum for CML resistance yielded 12 transfectant clones. Cosmids isolated from 7 of these clones conferred CML resistance when transfected into an independent, high-passage promastigote culture; at 12% human serum, the mean survival of transfectants was 37% (+/- 11.6%), and that of control transfectants was about 1%. Inserts within the 7 cosmids were unique. Determination of the complete DNA sequence for 1 cosmid indicated that its 32-kilobase insert was 89% identical (overall) to a 31-kilobase region of Leishmania major chromosome 36, which is predicted to encode 6 genes, all of which encode hypothetical proteins.

Cervical human papillomavirus infection and squamous intraepithelial lesions in rural Gambia, West Africa: viral sequence analysis and epidemiology

The development of effective strategies against cervical cancer in Africa requires accurate type specific data on human papillomavirus (HPV) prevalence, including determination of DNA sequences in order to maximise local vaccine efficacy. We have investigated cervical HPV infection and squamous intraepithelial lesions (SIL) in an unselected cohort of 1061 women in a rural Gambian community. Squamous intraepithelial lesions was diagnosed using cytology and histology, HPV was typed by PCR-ELISA of DNA extracts, which were also DNA sequenced. The prevalence of cervical HPV infection was 13% and SIL were observed in 7% of subjects. Human papillomavirus-16 was most prevalent and most strongly associated with SIL. Also common were HPV-18, -33, -58 and, notably, -35. Human papillomavirus DNA sequencing revealed HPV-16 samples to be exclusively African type 1 (Af1). Subjects of the Wolof ethnic group had a lower prevalence of HPV infection while subjects aged 25–44 years had a higher prevalence of cervical precancer than older or younger subjects. This first report of HPV prevalence in an unselected, unscreened rural population confirms high rates of SIL and HPV infection in West Africa. This study has implications for the vaccination of Gambian and other African populations in the prevention of cervical cancer.

LaNe RAGE: a new tool for genomic DNA flanking sequence determination

The determination of genomic DNA sequence flanking a known region is often problematic. Existing technologies depend on multiple, efficient enzyme-catalysed preparative processing steps and/or rely on relatively inefficient 'one-sided' PCR mechanisms. I demonstrate the application of a simple 'two-sided' PCR-based approach, lariat-dependent nested PCR for rapid amplification of genomic DNA ends (LaNe RAGE), applied to the mouse GAPDH and PGK1 gene flanking sequences. This demonstration offers great promise in applications such as genome walking, transposon mutagenesis mapping and DNA fingerprinting.