We previously developed short-term and long-term assays for autonomous replication of DNA in human cells. This study addresses the requirements for replication in these assays. Sixty-two random human genomic fragments ranging in size from 1 to 21 kb were cloned in a prokaryotic vector and tested for their replication ability in the short-term assay. We found a positive correlation between replication strength and fragment length, indicating that large size is favored for efficient autonomous replication in human cells. All large fragments replicated efficiently, suggesting that signals which can direct the initiation of DNA replication in human cells are either very abundant or have a low degree of sequence specificity. Similar results were obtained in the long-term assay. We also used the same assays to test in human cells a random series of fragments derived from Escherichia coli chromosomal DNA. The bacterial fragments supported replication less efficiently than the human fragments in the short-term and long-term assays. This result suggests that while the sequence signals involved in replication in human cells are found frequently in human DNA, they are uncommon in bacterial DNA.In this study we investigated the genetic requirements for origin of replication function in human cells. The study utilizes a system we have developed that allows us to test fragments of DNA for autonomous replication when introduced into human tissue culture cells in both long-and short-term assays. This system should ultimately permit us to deduce the DNA sequence requirements for autonomous replication. It is likely that this information will be relevant for chromosomal replication as well.Over the course of many cell divisions, extrachromosomal vectors are lost from the nuclei of mammalian cells unless they have a means of nuclear retention. The long-term replication assay that we developed overcomes the problem of plasmid loss by using a replication-defective vector derived from Epstein-Barr virus (EBV) that permits linked sequences to be retained in human cells (22). When random human DNA was cloned into this vector and introduced into human cells, we found that a large number of fragments could confer replication ability on the vector in a long-term assay. The resulting plasmids were stably maintained for at least 2 months in human cells.The fragments could also provide for replication in our short-term assay (22). In this assay, the fragments are moved to a prokaryotic vector that lacks all viral sequences, and DNA is harvested 4 days after transfection, since the plasmids have no means of nuclear retention. These experiments indicated that the human DNA fragments that we had isolated contained sequences involved in the initiation of DNA replication in human cells. Subsequent studies have shown that, like the chromosomes, these plasmids undergo replication once per cell cycle in human cells (unpublished data). This characteristic makes the autonomously replicating system a reasonable model for chromosomal replication.The hu...

PURPOSE Vietnam is undergoing rapid socio-economic transition with an increasing cancer burden. The contribution of modifiable risk factors to cancers in Vietnam has not been studied. Therefore, we sought to evaluate the attributable causes of cancer in Vietnam. METHODS We reviewed the data on burden of cancer in Vietnam from 2 cancer registries in Hanoi and Ho Chi Minh City between 1995 and 2012. Next, we calculated the fractions of cancers occurring in 2018 attributable to established modifiable risk factors whose impact could be quantified. Data on exposure prevalence were obtained for the period from 2000 to 2010 from national sources wherever possible. RESULTS Cancer incidence in Vietnam has decreased slightly in both sexes. Cancer related to infectious agents decreased sharply, whereas cancer related to nutrition and metabolism has increased. In 2018, established carcinogens included in the analysis explained 47.0% of cancer burden in Vietnam. Chronic infections accounted for 29.1% of cancers (34.7% in men and 22.1% in women), tobacco smoking for 13.5% (23.9% in men and 0.8% in women), and alcohol drinking for 10.3%. Passive smoking was responsible for 8.8% of cancers in women. Other risk factors, including overweight or obesity, nulliparity, and low vegetable and fruit intake, accounted for < 1% of all cancers each. CONCLUSION Cancer incidence is slowly decreasing in Vietnam, and the causes of more than half of cancers remain unexplained. This result underlines the need for further epidemiologic and fundamental research. Our findings confirm the notion that controlling oncogenic infections and decreasing tobacco smoking are the most effective approaches to reduce the burden of cancer in Vietnam, but other risk factors, including alcohol drinking and diet, should not be neglected.

We previously developed short-term and long-term assays for autonomous replication of DNA in human cells. This study addresses the requirements for replication in these assays. Sixty-two random human genomic fragments ranging in size from 1 to 21 kb were cloned in a prokaryotic vector and tested for their replication ability in the short-term assay. We found a positive correlation between replication strength and fragment length, indicating that large size is favored for efficient autonomous replication in human cells. All large fragments replicated efficiently, suggesting that signals which can direct the initiation of DNA replication in human cells are either very abundant or have a low degree of sequence specificity. Similar results were obtained in the long-term assay. We also used the same assays to test in human cells a random series of fragments derived from Escherichia coli chromosomal DNA. The bacterial fragments supported replication less efficiently than the human fragments in the short-term and long-term assays. This result suggests that while the sequence signals involved in replication in human cells are found frequently in human DNA, they are uncommon in bacterial DNA.

We studied the replication of random genomic DNA fragments from Saccharomyces cerevisiae in a long-term assay in human cells. Plasmids carrying large yeast DNA fragments were able to replicate autonomously in human cells. Efficiency of replication of yeast DNA fragments was comparable to that of similarly sized human DNA fragments and better than that of bacterial DNA. This result suggests that yeast genomic DNA contains sequence information needed for replication in human cells. To examine whether DNA replication in human cells would initiate specifically at a yeast origin of replication, we monitored initiation on a plasmid containing the yeast 2-micron autonomously replicating sequence (ARS) in yeast and human cells. We found that while replication initiates at the 2-micron ARS in yeast, it does not preferentially initiate at the ARS in human cells. This result suggests that the sequences that direct site specific replication initiation in yeast do not function in the same way in human cells, which initiate replication at a broader range of sequences.

Planktothrix agardhii is one of the freshwater cyanobacteria that can produce the hepatotoxin microcystins (MC)-a real threat to human and animal health. Knowledge of the biological role of MC in producing organisms is highly desired to understand the driving force of MC production. Recently, emerging evidences have suggested that MC may have protective role in cells facing environmental stress. If this is true, one should expect differences in the cellular protective mechanisms between MC-containing and MC-deficient mutant strains. To test this hypothesis, it would be essential to investigate the consequences of the loss of MC in Planktothrix in the transcriptional responses of its heat shock proteins (Hsps) to abiotic stresses-an important component of cellular stress response. However, a crucial first step is prerequisite for the isolation of hsp genes here, as the genome of Planktothrix has not been fully published. Therefore, we have successfully isolated four hsp genes including clpC (hsp100), htpG (hsp90), groEL (hsp60), and groES (hsp10) from Planktothrix agardhii PCC 7805 using ramped annealing PCR (RAN-PCR) with consensus-degenerate hybrid oligonucleotide primers (CODEHOP) and annealing control primer (ACP) system. In addition, some putative regulatory sequences found in the upstream region of groESL operon of Planktothrix agardhii were also discussed.