Long Stretches Of DNA Research Articles

In Silico Approaches Reveal the Potential for DNA Sequence-dependent Histone Octamer Affinity to Influence Chromatin Structure in Vivo

Nucleosome positioning signals embedded within the DNA sequence have the potential to influence the detailed structure of the higher-order chromatin fibre. In two previous studies of long stretches of DNA, encompassing the chicken beta-globin and ovine beta-lactoglobulin genes, respectively, we mapped the relative affinity of every site for the core histone octamer. In both cases a periodic arrangement of the in vitro positioning sites suggests that they might influence the folding of a nucleosome chain into higher-order structure; this hypothesis was borne out in the case of the beta-lactoglobulin gene, where the distribution of the in vitro positioning sites is related to the positions nucleosomes actually occupy in sheep liver cells. Here, we have exploited the in vitro nucleosome positioning datasets to simulate nucleosomal organisation using in silico approaches. We use the high-resolution, quantitative positioning maps to define a one-dimensional positioning energy lattice, which can be populated with a defined number of nucleosomes. Monte Carlo techniques are employed to simulate the behaviour of the model at equilibrium to produce a set of configurations, which provide a probability-based occupancy map. Employing a variety of techniques we show that the occupancy maps are a sensitive function of the histone octamer density (nucleosome repeat length) and find that a minimal change in this property can produce dramatic localised changes in structure. Although simulations generally give rise to regular periodic nucleosomal arrangements, they often show octamer density-dependent discontinuities, which tend to co-localise with sequences that adopt distinctive chromatin structure in vivo. Furthermore, the overall organisation of simulated chromatin structures are more closely related to the situation in vivo than is the original in vitro positioning data, particularly at a nucleosome density corresponding to the in vivo state. Although our model is simplified, we argue that it provides a unique insight into the influence that DNA sequence can have in determining chromatin structure and could serve as a useful basis for the incorporation of other parameters.

Combination of His-Tagged T4 Endonuclease VII with Microplate Array Diagonal Gel Electrophoresis for High-Throughput Mutation Scanning

Various physical mutation-scanning methods have been developed to avoid unnecessary resequencing of long stretches of DNA (1)(2)(3)(4)(5)(6). Protein-based mutation-scanning techniques include enzymatic digestion [reviewed in Ref. (7)], protein binding to a DNA duplex, and direct analyses of the in vivo or in vitro gene product. One such enzyme is T4 endonuclease VII (endoVII), the product of gene 49 of bacteriophage T4 (8). Radiolabel replacement with fluorescent tags has facilitated automated analysis (9). EndoVII recognizes heteroduplex structural distortions, nicking 2–6 bp 3′ to the distortion, with efficiency dependent on sequence context (10) and mismatch type(11). Perfectly matched DNA undergoes some background digestion, which produces a highly reproducible pattern (12). Mutation detection sensitivity obtained with endoVII digestion was found to be similar to that for denaturing HPLC and direct sequencing (13). Microplate array diagonal gel electrophoresis (MADGE) (14) provides an open-faced 96-well gel format for polyacrylamide gels. Recently, nondenaturing 192-, 384-, and 768-well formats of MADGE for high-throughput checking of PCR and post-PCR reactions (15) have been developed. We have combined, in proof-of-principle experiments, the mismatch digestion properties of endoVII with the high-throughput capabilities of MADGE and a newly developed denaturing MADGE format to create a simple mutation-scanning technique that can screen ∼1000 PCR samples during a single 35-min electrophoretic run. Plasmid pRB210 (T4 endonuclease VII in pET11a) was a kind gift from Professor B. Kemper (Institute for Genetics, University of Cologne, Germany). The PCR primers used to amplify the endoVII gene from pRB210 were as follows: forward, 5′-GCGCCATATGATGTTATTGAC-3′; reverse, 5′-CAGCGGATCCTCATTTTAAACT-3′. After trimming was performed with Bam HI and Nde I (New England Biolabs), pETendoVII was generated by ligation into pET15b (Novagen). Expressed N-terminal His-tagged endoVII was then purified by affinity chromatography. We used a single colony from pETendoVII-transfected BL21 …

Phillip Sharp—Nobel Prize for Discovery of “Split Genes”

American molecular biologist Phillip Allen Sharp received the 1993 Nobel Prize in physiology or medicine for his discovery of “split genes.” He found that these genes are the most common type of gene structure in higher organisms, including humans. He shared the prize with Richard John Roberts (1943-), who discovered split genes independently of Sharp. The discovery of split genes has been of fundamental importance to basic research in biology as well as medical research on the development of cancer and other diseases. The discovery of split genes led to the prediction of the genetic process of splicing. Sharp was born on June 6, 1944, in Falmouth, Ky, on a small farm where his parents grew tobacco and corn. He attended Union College in Barbourville in southeastern Kentucky and received his BA degree in 1966. He subsequently enrolled at the University of Illinois in Champaign-Urbana and earned a PhD degree in chemistry in 1969. He was a postdoctoral fellow at the California Institute of Technology in Pasadena from 1969 to 1971. In 1971, Sharp became a member of the faculty of the Cold Spring Harbor Laboratory in southeastern New York State, where he worked (from 1971 to 1972) with 1962 Nobel laureate James Watson (1928-). From 1972 to 1974, Sharp was a senior research investigator at the laboratory. Sharp left Cold Spring Harbor Laboratory in 1974 to join the faculty at the Massachusetts Institute of Technology (MIT) in Cambridge, where his prize-winning research was performed. In the 1980s and the early 1990s, he was associate director and then director of the MIT Center for Cancer Research. In 1991, he was appointed head of the Department of Biology at MIT, and in 1992, he became the first Salvador E. Luria (1912-1991) Professor, a chair established at MIT to honor the 1969 Nobel laureate. In 1977, Sharp and his colleagues discovered that a single molecule of messenger RNA (mRNA) from an adenovirus corresponds to 4 separate discontinuous segments of DNA. (The segments of DNA that code for proteins [exons] are separated by long stretches of DNA [introns] that do not contain genetic information; the terms exon and intron were coined by Walter Gilbert [1932-], the 1980 Nobel laureate in chemistry.) Working independently of Sharp, Richard Roberts and his research team made the same discovery. Previously, biologists had believed that genes were continuous stretches of DNA that served as direct templates for mRNA in the assembly of proteins. In addition to the Nobel Prize, Sharp has received many honors and awards, including the Eli Lilly Award (1980), the Ricketts Award (1985), the Sloan Prize (1986), the Gairdner Foundation Award (1986), the Horwitz Prize (1988), the Lasker Award (1988), and the Dickson Prize (1990). He was honored on a stamp issued by the Palau Islands in 2000.

Geographic stratification of linkage disequilibrium: a worldwide population study in a region of chromosome 22

Recent studies of haplotype diversity in a number of genomic regions have suggested that long stretches of DNA are preserved in the same chromosome, with little evidence of recombination events. The knowledge of the extent and strength of these haplotypes could become a powerful tool for future genetic analysis of complex traits. Different patterns of linkage disequilibrium (LD) have been found when comparing individuals of African and European descent, but there is scarce knowledge about the worldwide population stratification. Thus, the study of haplotype composition and the pattern of LD from a global perspective are relevant for elucidating their geographical stratification, as it may have implications in the future analysis of complex traits. We have typed 12 single nucleotide polymorphisms in a chromosome 22 region--previously described as having high LD levels in European populations -- in 39 different world populations. Haplotype structure has a clear continental structure with marked heterogeneity within some continents (Africa, America). The pattern of LD among neighbouring markers exhibits a strong clustering of all East Asian populations on the one hand and of Western Eurasian populations (including Europe) on the other, revealing only two major LD patterns, but with some very specific outliers due to specific demographic histories. Moreover, it should be taken into account that African populations are highly heterogeneous. The present results support the existence of a wide (but not total) communality in LD patterns in human populations from different continental regions, despite differences in their demographic histories, as population factors seem to be less relevant compared with genomic forces in shaping the patterns of LD.

Relative efficiency of ambiguous vs. directly measured haplotype frequencies.

Haplotypes are useful for both fine-mapping of susceptibility loci and evaluation of sequence variation at multiple sites along a chromosome. However, they are difficult to directly measure over long stretches of DNA in diploid organisms. Consequently, multiple genetic markers are typically measured, without linkage phase information, giving rise to a subject's diplotype. From diplotype data, haplotypes are often inferred by pedigree information, or treated as partially missing data when haplotype frequencies are estimated among unrelated subjects. This latter ambiguity can increase the variance of the estimated haplotype frequencies. Douglas et al. ([2001] Nat. Genet. 28:361-364) recently quantified the relative efficiency of estimating haplotype frequencies from the diplotypes of unrelated subjects, relative to directly measured haplotypes via somatic cell hybrids (conversion technology), and demonstrated that unknown linkage phase can lead to a large loss of efficiency. However, their results were based on linkage equilibrium among marker loci, which may not be realistic for closely linked markers. We extend their relative efficiency calculations by several aspects: 1) allowance for linkage disequilbrium (LD) among marker loci; 2) evaluation of different patterns of LD; and 3) evaluation of nuclear families with and without parents. We show that although the loss in efficiency of haplotype frequencies among unrelated subjects decreases as LD increases to its maximum value, the general conclusions of Douglas et al. ([2001] Nat. Genet. 28:361-364) hold true for a variety of LD patterns and magnitudes. However, our results also demonstrate that trios of parents+one child are highly efficient for haplotype frequency estimation, that additional children offer little information, and that siblings without parents can be grossly inefficient. Genet. Epidemiol. 23:426-443, 2002.

Global regulation of virulence determinants in Staphylococcus Aureus by the SarA protein family

In S. aureus, the production of virulence determinants such as cell wall adhesins and exotoxins during the growth cycle is controlled by global regulators such as SarA and agr. Genomic scan reveals 16 two-component regulatory systems (e.g. agr and sae) as well as a family of SarA homologs in S. aureus. We call the SarA homologs the SarA protein family. Many of the members in this protein family are either small basic proteins (<153 residues) or two-domain proteins in which a single domain shares sequence similarity to each of the small basic proteins. Recent crystal structures of SarR and SarA reveal dimeric structures for these proteins. Because of its structure and unique mode of DNA binding, SarR, and possibly other SarA family members, may belong to a new functional class of the winged-helix family, accommodating long stretch of DNA with bending points. Based on sequence homology, we hypothesize that the SarA protein family may entail homologous structures with similar DNA-binding motifs but divergent activation domains. An understanding of how these regulators interact with each other in vivo and how they sense environmental signals to control virulence gene expression (e.g. alpha-hemolysin) will be important to our eventual goal of disrupting the regulatory network.

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic poleta.

Phylogenetic analysis of Y-family DNA polymerases suggests that it can be subdivided into several discrete branches consisting of UmuC/DinB/Rev1/Rad30/Rad30A and Rad30B. The most diverse is the DinB family that is found in all three kingdoms of life. Searches of the complete genome of the crenarchaeon Sulfolobus solfataricus P2 reveal that it possesses a DinB homolog that has been termed DNA polymerase IV (Dpo4). We have overproduced and purified native Dpo4 protein and report here its enzymatic characterization. Dpo4 is thermostable, but can also synthesize DNA at 37 degrees C. Under these conditions, the enzyme exhibits misinsertion fidelities in the range of 8 x 10(-3) to 3 x 10(-4). Dpo4 is distributive but at high enzyme to template ratios can synthesize long stretches of DNA and can substitute for Taq polymerase in PCR. On damaged DNA templates, Dpo4 can facilitate translesion replication of an abasic site, a cis-syn thymine-thymine dimer, as well as acetyl aminofluorene adducted- and cisplatinated-guanine residues. Thus, although phylogenetically related to DinB polymerases, our studies suggest that the archaeal Dpo4 enzyme exhibits lesion-bypass properties that are, in fact, more akin to those of eukaryotic poleta.

How to proteins move along DNA? Lessons from type-I and type-III restriction endonucleases.

Protein-mediated communications on DNA are universally important. The translocation of DNA driven by a high-energy phosphoryl potential allows long stretches of DNA to be traversed without dissociation. Type-I and type-III enzymes both use a common DNA-tracking mechanism to move along DNA, dependent on the hydrolysis of ATP. Type-I enzymes cleave DNA at distant DNA sites (and in some cases close to the site), due to a stall in enzyme motion. This can be due to collision with another translocating type-I enzyme or, on circular DNA, due to an increased topological load. ATP hydrolysis is considerable, and continues after DNA cleavage. Type-III enzymes only cleave DNA proximal to their sites due to collision between two endonucleases tracking with defined polarity. ATP hydrolysis is less than with the type-I enzymes. Homology to DNA helicases has been found within the HsdR and Res subunits. Mutagenesis of the DEAD-box motifs affects both ATP hydrolysis and DNA cleavage. This demonstrates a tight link between ATPase and endonuclease activities. A strand-separation mechanism akin to the DNA helicases is a possibility. The DNA-based motor proteins are mechanistically ill-defined. Further study using some of the techniques pioneered with classical motor proteins will be needed to reveal more detail.

Distinguishing recombination and intragenic gene conversion by linkage disequilibrium patterns.

Deterministic theory suggests that reciprocal recombination and intragenic, interallelic conversion have different effects on the linkage disequilibrium between a pair of genetic markers. Under a model of reciprocal recombination, the decay rate of linkage disequilibrium depends on the distance between the two markers, while under conversion the decay rate is independent of this distance, provided that conversion tracts are short. A population genetic three-locus model provides a function Q of two-locus linkage disequilibria. Viewed as a random variable, Q is the basis for a test of the relative impact of conversion and recombination. This test requires haplotype frequency data of a sufficiently variable three-locus system. One of the few examples currently available is data from the Human Leukocyte Antigen (HLA) class I genes of three Amerindian populations. We find that conversion may have played a dominant role in shaping haplotype patterns over short stretches of DNA, whereas reciprocal recombination may have played a greater role over longer stretches of DNA. However, in order to draw firm conclusions more independent data are necessary.

Mapping undetected mutations within a gene-evidence for two preferential regions in the DMD gene.

A maximum-likelihood method is developed to estimate the frequency distribution of undetected mutations (presumably point mutations, small deletions, insertions) along a gene, where the gene extends over a long stretch of DNA. In each family, the point of the mutation is potentially at a different location within the gene. In this sense, there is genetic heterogeneity among families and the method estimates the proportion of families whose mutation is at (or in the vicinity of) a given point inside the gene. Our method is applied to a sample of 75 families with Duchenne muscular dystrophy in which the disease mutation remained undetected. We find two preferential regions for these undetected mutations, with an estimated 85% of families having their mutation in one region and the remaining 15% of families in the other. The new method is expected to be useful in finding small mutations in any of the currently known large genes.

Construction and characterization of a Nicotiana plumbaginifolia genomic library in a yeast artificial chromosome

Large-size DNA of Nicotiana plumbaginifolia was manipulated in solution, concentrated by 2-butanol and cloned into a yeast artificial chromosome, pYACCMC5. A YAC library consisting of 22 000 clones with an average insert size of 190 kb has been constructed. The sizes of YAC inserts in this library ranged from 80–660 kb, indicating that 2-butanol can be used for concentrating large-size DNA with minimum shearing damage. This library represents 84% of the genome of N. plumbaginifolia. Characterization of this library revealed that only 6% of clones in the library contained sequences derived from chloroplast DNA. Some YAC inserts contained relatively long stretches of DNA, 100–180 kb, uninterrupted with repetitive sequences. Furthermore, both ends of most DNA molecules cloned in pYACCMC5 can be reisolated efficiently as recombinant plasmids in Escherichia coli. Approximately one third of the subcloned end fragments contained low- or single-copy DNA sequences. Single-copy end fragments are valuable probes to identify overlapping YAC clones by chromosome walking. The method for concentrating large-size DNA and the vector for YAC cloning described here should be useful in genome research programs. The YAC library presented here will facilitate mapping and cloning of genes from the genome of N. plumbaginifolia, which has been used for over a decade as a model species in plant cell genetics.

Nucleic acid crystallography: a view from the nucleic acid database.

What are the future directions of the field of nucleic acid crystallography? Although there have been many duplex structures determined, the sample is still relatively small. This is especially true if one wants to derive enough information about the relationships between sequence and structure. Indeed, there are data for all the possible 10 dimer steps, but for some steps it is very limited. If the structural code resides in trimers or tetrad steps then there is simply not enough data to do meaningful statistical analyses. So the first direction that needs to be explored is the determination of more structures with more varied sequences. The other noticeable thing about the data is the shortness of the strands. While it is probably true that attempts to crystallize very long sequences will not meet with success, the idea of crystallizing sequences engineered to fit together via sticky ends such as has been done for the CAP-DNA complex (Schultz et al., 1990) should give data about the behavior of much longer stretches of DNA. The question of the effects of environment on the structure of DNA continues to be a very important one to address since DNA is rarely alone. The preliminary data we have analysed from the current sample shows that the conformation of some steps are very sensitive to packing type. Numerous studies of the hydration around DNA shows that there is a real synergy between the hydration structure and the base conformation. More data will allow further quantitation of these observations. RNA structure is the next very exciting frontier. The emerging structures of duplexes with internal loops, the two hammerhead ribozyme structures and the group I intron ribozyme have given us a glimpse of the complexity and elegance of this class of molecules. With the technology now in place to allow the determination of the structures of these molecules, the expectation is that now we will see a large increase in the number of these structures in the NDB.

Chromatin structure mapping in vivo using methyltransferases

This chapter discusses the mapping of chromatin structure in vivo using methyltransferases. Expression of prokaryotic methyltransferases in Saccharomyces cerevisiae (S. cerevisiae), the genome of which contains no endogenous detectable methylation is used as a means of probing chromatin structure in living cells. A DNA fragment from E. coli containing the dam methyltransferase gene was introduced into the LYS2 locus of S. cerevisiae by transfection. The gene was expressed from a fortuitous promoter in an uncontrolled fashion, leading to the modification of adenines in the GATC sequence recognized by this methyltransferase. These studies were seminal in use of such enzymes as probes for chromatin structure in living cells. The use of a positive chemical method for the detection of modified residues under conditions of limited overall modification allows quantitative analysis of accessibility over long stretches of DNA. Although currently only demonstrated in S. cerevisiae, the general approaches outlined in this chapter should be portable to some other species for analysis of chromatin structure. A limitation in use for other species is the occurrence of endogenous CG cytidine methylation. Clearly the sites modified in normal cells are detected in the chemical analysis. Similar, although less dramatic, problems occur for GATC methylation, where one cytidine in four will occur in the sequence GATCG. For this reason, as well as enhancement of the level of resolution of mapping, an active search is underway for cytidine methyltransferases that recognize di- or trinucleotides that occur with higher frequency than CG.

Isolation and characterisation of dhel II, a DNA helicase from Drosophila melanogaster embryos stimulated by Escherichia coli-type single-stranded-DNA-binding proteins.

We have purified a DNA helicase from Drosophila embryos by following unwinding activity during the purification of the cellular single-stranded DNA-binding protein dRP-A. This DNA helicase unwinds DNA 5' to 3', has a salt-tolerant activity, and has a preference for purine triphosphates as cofactors for the unwinding reaction. The purified enzyme consists of a single polypeptide of 120 kDa, which cosediments with the helicase activity. Sedimentation analysis suggests that this polypeptide exists as a monomer under high and low salt conditions. Dhel II is able to unwind long stretches of DNA, but with decreased efficiency. Addition of Escherichia coli-like single-stranded DNA-binding proteins stimulates the unwinding activity at least 10-fold on substrates greater than 200 nucleotides. In particular, the mitochondrial single-stranded DNA-binding protein isolated from Drosophila embryos is able to stimulate unwinding by dhel II. These properties show that the helicase described is different from another Drosophila helicase dhel I; it has thus has been classified as dhel II.

Monomeric RecBCD Enzyme Binds and Unwinds DNA

RecBCD enzyme is a multifunctional nuclease that is essential for the major pathway of homologous genetic recombination in Escherichia coli. It has a potent helicase activity that uses ATP hydrolysis to unwind very long stretches of DNA. The functional form of RecBCD enzyme has been unclear, since M(r) of 250,000-655,000 have been previously reported. We have isolated two oligomeric forms of the enzyme, one (monomeric) containing a single copy of the RecB, RecC, and RecD polypeptides, and the other (dimeric) containing two copies of each polypeptide. We show here that the monomeric form of the enzyme (M(r) approximately 330,000) can form a stable initiation complex on the end of ds DNA. Depending on the nature of the ds end, KD estimates ranged from approximately 0.1 nM to approximately 0.7 nM in the presence of Mg2+ ions, which enhanced but was not required for binding. We further showed that the complex of monomeric RecBCD enzyme and a ds DNA end was competent to unwind DNA. A general model for the action of helicases has been proposed that uses repeated conformational changes between two states of a complex between DNA and a dimeric form of the enzyme. Our results make such a model unlikely for RecBCD enzyme.

Characterization of RecA mediated homologous pairing on nitrocellulose membrane

Reactions between a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) provide an efficienty model to study RecA promoted homologous recombination. We have devised an assay in which the ssDNA is first bound to a nitrocellulose membrane. RecA protein is loaded on this membrane (loading step) which is then incubated with a labelled homologous dsDNA (incubation step). Since this assay can be used for study of mutant RecA proteins or RecA-like activities in crude extracts from other organisms, we have characterized the reaction promoted on the membrane. Under these new conditions, the reaction keeps the main characteristics observed with classical assays performed in solution: increasing NaCl concentration destabilized the RecA-DNA complex; ATPγS was required for formation of stable RecA-DNA complex, initiation of the reaction exhibits the same polarity as in classical assays, a complete strand exchange with a 44 bp long duplex oligonucleotide has been recorded under our conditions. Moreover, our results indicate that the binding of RecA protein itself to the nitrocellulose membrane did not impair its ability to promote homologous pairing. Pairing reactions involving long dsDNA (6407 bp) were more efficient with hydrolysable ATP than with ATPγS only when the ssDNA was bound to the membrane. Furthermore, ATP hydrolysis was not required when using short dsDNA (44 bp). These results constitute experimental support for a new role for the ATPase activity of RecA protein: the energy produced could favor the initiator of RecA mediated recombination involving long stretches of DNA which have restricted freedom to rotation.

The extreme C terminus of herpes simplex virus DNA polymerase is crucial for functional interaction with processivity factor UL42 and for viral replication.

The herpes simplex virus DNA polymerase is composed of two subunits, a large catalytic subunit (Pol) and a smaller subunit (UL42) that increases the processivity of the holoenzyme. The interaction between the two polypeptides is of interest both for the mechanism by which it enables the enzyme to synthesize long stretches of DNA processively and as a possible target for the rational design of novel antiviral drugs. Here, we demonstrate through a combination of insertion and deletion mutagenesis that the carboxy-terminal 35 amino acids of Pol are crucial for binding UL42. The functional importance of the interaction was confirmed by the finding that a pol mutant defective for UL42 binding retained polymerase activity, but did not synthesize longer DNA products in the presence of UL42. Moreover, several association-incompetent mutants failed to complement the replication of a pol null mutant in a transient transfection assay, confirming that the Pol-UL42 interaction is necessary for virus replication in vivo and therefore a valid target for directed drug design.

Complexity in the major histocompatibility complex.

The human major histocompatibility complex (MHC) is one of the most intensively studied regions of the human genome, containing over 70 known genes and spanning about 4 million base pairs (4 Mbp) of DNA on chromosome 6p21.3 (Klein, 1986). It can be divided up into three regions: the class I region (telomeric), the class II region (centromeric), and the class III region (between class I and II), which includes the complement component genes C2, C4, and Bf (Trowsdale & Campbell, 1988). The MHC has been mapped in detail using pulse field gel electrophoresis (PFGE) and by cloning in yeast artificial chromosome (YAC) and cosmid vectors, revealing long stretches of DNA between the regions as well as between individual class I and class II genes. Novel genes, that have no sequence relationships with class I, class II or complement components, have recently been found in these areas, and we will present an update on these after reviewing the more established loci.

The DNA synthesizing subunit of polymerase-primase from calf thymus.

The DNA synthesizing subunit (alpha-subunit) of DNA polymerase-alpha from calf thymus was separated from the other three subunits by immunoaffinity chromatography. The enzymatic properties of the alpha-subunit were characterized and compared with those of the four-subunit complex. Free alpha-subunit behaved in many respects like the four-subunit polymerase-primase. It was inhibited by aphidicolin and butylanilino-deoxyATP and catalyzed DNA synthesis on both gapped duplex DNA as well as primed single-stranded DNA with a preference of gapped DNA. The alpha-subunit is a quasi-processive enzyme with a processivity for about 9 nucleotides incorporated per single primer binding event. This is 2-fold lower than the processivity of the four-subunit complex. Despite this moderate processivity, free alpha-subunit was able to synthesize long stretches of DNA on singly primed natural psi X174am16 DNA. The accuracy of DNA synthesis of the free alpha-subunit was determined by using the psi X174am16 reversion assay to be 1 error per 50,000 nucleotides incorporated. An in vitro accuracy of 1 error in 54,000 nucleotides incorporated was measured in parallel for the four-subunit complex. Thus, the smaller subunits do not contribute to the overall accuracy of DNA polymerase-alpha. Consistent with this result is the observation that the polymerase to 3'----5'-exonuclease ratio was less than 1 to 2,500,000. Therefore, there is no evidence for the action of a cryptic proofreading activity with the alpha-subunit of DNA polymerase-alpha of mammalian origin.

Origins of the human genome project

The Human Genome Project has become a reality. Building on a debate that dates back to 1985, several genome projects are now in full stride around the world, and more are likely to form in the next several years. Italy began its genome program in 1987, and the United Kingdom and U.S.S.R. in 1988. The European communities mounted several genome projects on yeast, bacteria, Drosophila, and Arabidospis thaliana (a rapidly growing plant with a small genome) in 1988, and in 1990 commenced a new 2-year program on the human genome. In the United States, we have completed the first year of operation of the National Center for Human Genome Research at the National Institutes of Health (NIH), now the largest single funding source for genome research in the world. There have been dedicated budgets focused on genome-scale research at NIH, the U.S. Department of Energy, and the Howard Hughes Medical Institute for several years, and results are beginning to accumulate. There were three annual meetings on genome mapping and sequencing at Cold Spring Harbor, New York, in the spring of 1988, 1989, and 1990; the talks have shifted from a discussion about how to approach problems to presenting results from experiments already performed. We have finally begun to work rather than merely talk. The purpose of genome projects is to assemble data on the structure of DNA in human chromosomes and those of other organisms. A second goal is to develop new technologies to perform mapping and sequencing. There have been impressive technical advances in the past 5 years since the debate about the human genome project began. We are on the verge of beginning pilot projects to test several approaches to sequencing long stretches of DNA, using both automation and manual methods. Ordered sets of yeast artificial chromosome and cosmid clones have been assembled to span more than 2 million base pairs of several human chromosomes, and a region of 10 million base pairs has been assembled for Caenorhabditis elegans by a collaboration between Washington University and the Medical Research Council laboratory in Cambridge, U.K. This project is now turning to sequencing C. elegans DNA as a logical extension of this work. These are but the first fruits of the genome project. There is much more to come.