A simple method for DNA extraction from marine bacteria that produce extracellular materials

We present a simple method for extracting DNA from the marine bacteria Hahella chejuensis, a Streptomyces sp., and a Cytophaga sp. Previously, DNA purification from these strains was hindered by the presence of extracellular materials. In our extraction method, the marine bacteria are lysed by freezing and grinding in liquid nitrogen, and treated with SDS. The extracted DNA is purified using a phenol/chloroform mixture, and precipitated in isopropanol. The extracted DNA is of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion, genomic DNA blot hybridization, and genomic DNA library construction. We used this method to extract genomic DNA from several other marine bacteria. Our method is a reproducible, simple, and rapid technique for routine DNA extractions from marine bacteria. Furthermore, the low cost of this method makes it attractive for large-scale studies.     

Characterisation of a repetitive DNA family from Entamoeba histolytica containing Saccharomyces cerevisiae ARS consensus sequences

Several repetitive DNA families were identified in Entamoeba histolytica DNA digested with Sau3AI. Characterisation of one of these repetitive DNA families showed the presence of multiple copies of Saccharomyces cerevisiae autonomously replicating sequence (ARS) core consensus sequences. The E. histolytica ARS consensus sequences allowed a yeast-integrating plasmid, YIP5, to replicate autonomously in S. cerevisiae. A 'bent DNA' fragment was located in one member of this E. histolytica repetitive DNA family.     

Direct cloning of specific genomic DNA sequences in plasmid libraries following fragment enrichment.

We describe a simple method to directly clone any DNA fragment for which a flanking restriction enzyme map is known. Genomic DNA is digested with multiple enzymes cutting outside the fragment to be cloned, selected by electroelution from an agarose gel, and cloned directly into a plasmid vector. It is only necessary to screen 10-1000 colonies and recombinant DNA is ready for immediate molecular analysis without further subcloning. The use of this technique is demonstrated for the cloning of a sequence from within the human alpha-globin complex that was previously shown to be "unclonable" in bacteriophage and cosmid vectors and which is a multiallelic general genetic marker, as well as both beta-globin alleles from an individual with beta-thalassaemia.     

Analysis of fragment homology among DNA sequences from cytochrome P450 families 4 and 6

Cytochrome P450s comprise a diverse superfamily of proteins that often share as little as 12% amino acid identity. Accordingly, the identification of novel gene families, subfamilies and alleles has been based primarily on ‘benchmark’ levels of global amino acid identity and, more recently, phylogenetic relatedness has been used to resolve ambiguous relationships. However, PCR-based cloning strategies have resulted in a large increase in the number of short DNA sequences, particularly among insects. Many of these fragments remain unnamed and even their gene family membership remains unknown due to the uncertainty as to whether these fragments accurately reflect the levels of sequence identity or patterns of evolutionary divergence exhibited by groups of full-length P450 sequences. As a result, the nature of P450 diversity among insects remains obscure. In this paper, P450 sequences belonging to the two major gene families, that is, families CYP4 and CYP6, are analyzed by comparing segments to full-length sequences. A parameter called the Segmental Divergence Index is used to characterize segments of P450s with respect to the degree to which they mirror the divergence of full-length sequences. The reliability of these fragments in phylogenetic analyses is also tested. The results of these analyses support the use of some commonly amplified P450 gene fragments for naming new P450 genes and for studies of P450 diversity and evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alignment editing and identification of consensus secondary structures for nucleic acid sequences: interactive use of dot matrix representations.

We present a computer-aided approach for identifying and aligning consensus secondary structure within a set of functionally related oligonucleotide sequences aligned by sequence. The method relies on visualization of secondary structure using a generalization of the dot matrix representation appropriate for consensus sequence data sets. An interactive computer program implementing such a visualization of consensus structure has been developed. The program allows for alignment editing, data and display filtering and various modes of base pair representation, including co-variation. The utility of this approach is demonstrated with four sample data sets derived from in vitro selection experiments and one data set comprising tRNA sequences.     

Nucleotide, dinucleotide and trinucleotide frequencies explain patterns observed in chaos game representations of DNA sequences.

The chaos game representation (CGR) is a scatter plot derived from a DNA sequence, with each point of the plot corresponding to one base of the sequence. If the DNA sequence were a random collection of bases, the CGR would be a uniformly filled square; conversely, any patterns visible in the CGR represent some pattern (information) in the DNA sequence. In this paper, patterns previously observed in a variety of DNA sequences are explained solely in terms of nucleotide, dinucleotide and trinucleotide frequencies.     

DNA polymerase delta: gene sequences from Plasmodium falciparum indicate that this enzyme is more highly conserved than DNA polymerase alpha.

Genes encoding proteins homologous to the catalytic subunits of DNA polymerase alpha and delta have been cloned from the human malaria parasite Plasmodium falciparum. These are among the first cellular replicative DNA polymerase genes to be cloned and their sequences allow us to make new statements about the relative degrees of conservation of these two enzymes. The most important finding was that P. falciparum Pol delta showed considerable homology to the only other Pol delta enzyme for which published sequence is available, that of S. cerevisiae, displaying an overall amino acid identity of 45% and identity over a highly conserved central region of 59%. In contrast, the level of identity shown over the equivalent central region of Pol alpha between the P. falciparum and S. cerevisiae sequences is only 32%. The sequence data also allowed us to examine the degree of conservation in putative exonuclease domains of Pol delta. The Pol delta gene of P. falciparum maps to chromosome 10 and evidence is presented for the presence of different sized Pol delta mRNA's in the asexual and sexual erythrocytic stages of parasite development.     

Binding of the Escherichia coli cyclic AMP receptor protein to DNA fragments containing consensus nucleotide sequences.

Binding of the Escherichia coli CRP protein to DNA fragments carrying nucleotide sequences closely corresponding to the consensus is very tight with a dissociation time of over 2 h in our conditions. The concentration of cyclic AMP required for this binding is below the physiological range of intracellular cyclic AMP concentrations. Changes in nucleotide sequence at positions that are not well-conserved between different naturally-occurring CRP sites allow a more rapid dissociation of CRP-DNA complexes. There is an inverse correlation between the stability of CRP binding to sites in vitro and the repression by glucose of expression dependent on these sites in vivo: expression that is dependent on the tighter binding sites cannot be repressed by the inclusion of glucose in the growth medium.     

Properties of some monkey DNA sequences obtained by a procedure that enriches for DNA replication origins.

Twelve clones of monkey DNA obtained by a procedure that enriches 10(3)- to 10(4)-fold for nascent sequences activated early in S phase (G. Kaufmann, M. Zannis-Hadjopoulos, and R. G. Martin, Mol. Cell. Biol. 5:721-727, 1985) have been examined. Only 2 of the 12 ors sequences (origin-enriched sequences) are unique (ors1 and ors8). Three contain the highly reiterated Alu family (ors3, ors9, and ors11). One contains the highly reiterated alpha-satellite family (ors12), but none contain the Kpn family. Those remaining contain middle repetitive sequences. Two examples of the same middle repetitive sequence were found (ors2 and ors6). Three of the middle repetitive sequences (the ors2-ors6 pair, ors5, and ors10) are moderately dispersed; one (ors4) is highly dispersed. The last, ors7, has been mapped to the bona fide replication origin of the D loop of mitochondrial DNA. Of the nine ors sequences tested, half possess snapback (intrachain reannealing) properties.     

A cloned fragment of HeLa DNA containing consensus sequences of satellite II and III DNA hybridizes with the Drosophila P-element and with the 1.8 kb family of human KpnI fragments

We have cloned a repetitive EcoRI fragment from the human genome which displays weak homologies with the Drosophila melanogaster transposable P-element. This cloned DNA appeared not to be a mobile element but, instead, a divergent member of human satellite II or III DNAs. We present here the first complete nucleotide sequence of a 1.797 kilobase pair (kb) satellite-like DNA. Moreover, this EcoRI satellite monomer contains a unique sequence of 49 basepairs (bp) that is devoid of the satellite consensus repeat 5'TTCCA3'. Southern hybridization analysis revealed that the cloned insert is closely related to a highly repetitive 1.8 kb KpnI family of tandemly organized satellite DNAs. Thus, the relationships among these satellite DNA families appear to be complex and may be a factor in their copy number, position and spatial organization.     

Rigorous pattern-recognition methods for DNA sequences. Analysis of promoter sequences from Escherichia coli

The basic nature of the sequence features that define a promoter sequence for Escherichia coli RNA polymerase have been established by a variety of biochemical and genetic methods. We have developed rigorous analytical methods for finding unknown patterns that occur imperfectly in a set of several sequences, and have used them to examine a set of bacterial promoters. The algorithm easily discovers the "consensus" sequences for the -10 and -35 regions, which are essentially identical to the results of previous analyses, but requires no prior assumptions about the common patterns. By explicitly specifying the nature of the search for consensus sequences, we give a rigorous definition to this concept that should be widely applicable. We also have provided estimates for the statistical significance of common patterns discovered in sets of sequences. In addition to providing a rigorous basis for defining known consensus regions, we have found additional features in these promoters that may have functional significance. These added features were located on either side of the -35 region. The pattern 5', or upstream, from the -35 region was found using the standard alphabet (A, G, C and T), but the pattern between the -10 and the -35 regions was detectable only in a sub-alphabet. Recent results relating DNA sequence to helix conformation suggest that the former (upstream) pattern may have a functional significance. Possible roles in promoter function are discussed in this light, and an observation of altered promoter function involving the upstream region is reported that appears to support the suggestion of function in at least one case.     

Complete mitochondrial DNA genome sequences show that modern birds are not descended from transitional shorebirds

To test the hypothesis put forward by Feduccia of the origin of modern birds from transitional birds, we sequenced the first two complete mitochondrial genomes of shorebirds (ruddy turnstone and blackish oystercatcher) and compared their sequences with those of already published avian genomes. When corrected for rate heterogeneity across sites and non-homogeneous nucleotide compositions among lineages in maximum likelihood (ML), the optimal tree places palaeognath birds as sister to the neognaths including shorebirds. This optimal topology is a re-rooting of recently published ordinal-level avian trees derived from mitochondrial sequences. Using a penalized likelihood (PL) rate-smoothing process in conjunction with dates estimated from fossils, we show that the basal splits in the bird tree are much older than the Cretaceous-Tertiary (K-T) boundary, reinforcing previous molecular studies that rejected the derivation of modern birds from transitional shorebirds. Our mean estimate for the origin of modern birds at about 123 million years ago (Myr ago) is quite close to recent estimates using both nuclear and mitochondrial genes, and supports theories of continental break-up as a driving force in avian diversification. Not only did many modern orders of birds originate well before the K-T boundary, but the radiation of major clades occurred over an extended period of at least 40 Myr ago, thus also falsifying Feduccia's rapid radiation scenario following a K-T bottleneck.     

DNA from eukaryotic cells contains unusually long pyrimidine sequences.

Analysis of DNA samples from several different organisms revealed the presence of unexpectedly long pyrimidine tracts (polypyrimidine), ranging in size from about 25 to 200 nucleotides in length. This fraction of the DNA accounted for 0.07-0.8 percent of the total thymidine residues. Pyrimidine clusters of this length and amount would not be expected on a random basis, suggesting they may have been conserved during evolution because they are required for a specialized function(s).     

变形链球菌DNA酶切片段在大肠杆菌中克隆的研究

将变链菌染色体DNA和pBR322 DNA,分别用HindⅢ酶切,再用T_4DNA连接酶连接,转化到大肠杆菌R_5受体菌中。根据插入灭活的原理,筛选Ap~r、Tc~s菌株,并经抗药性、琼脂糖凝胶电泳,酶切、核酸杂交及再转化等试验证明,已将变链菌DNA酶切片段克隆到大肠杆菌中,获得2627个克隆株,建立了基因库。为筛选变链菌保护性抗原基因,进一步研制基因工程龋齿菌苗奠定了基础。