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PubMed1996

Inferring relatedness of a macromolecule to a sequence database without sequencing.

Derivation of biological information of a macromolecule isolate based on sequence similarity is playing a significant role in numerous areas of biological research. However, it is often the case that a researcher obtains more macromolecule isolates than can be sequenced practically, due either to the high cost of sequencing or lack of specialized equipment and personnel. To overcome this difficulty, we study the problem of obtaining biological information (such as sequence information) about a macromolecule isolate using only (i) the fragmentation pattern of that isolate obtained from digestion with enzymes and (ii) a database D of sequences. We investigate a three phase approach to solving this problem. In the first phase, we obtain a restriction pattern of the isolate while analytically deriving the corresponding restriction maps of the sequences in the database. In the second phase, we identify a set S [symbol: see text] D of sequences which have restriction maps that are most similar to the unknown isolate's restriction pattern. This task is complicated by the fact that we have only approximate fragment lengths for the unknown isolate and that we do not know the actual ordering of the unknown isolate's fragments. Despite these difficulties, we derive experimental results which indicate maximum matching techniques are effective in identifying the correct set most of the time. In the third phase, we use the set S to infer biological information (such as sequence information or hierarchical classification information) about the unknown isolate. We demonstrate experimentally that the closeness of the sequences in the set S to each other can be used to infer the relatedness of the unknown isolate to the sequences of the set S. Furthermore, the confidence of this inferred information is strongly correlated to the minimum pairwise relatedness of any two elements in S.

Databases, Factual↗
PubMed1990

The gdhA gene is located at 38.6 minutes on the Escherichia coli map.

We report here that the gdhA gene of Escherichia coli, which encodes the NADP-specific glutamate dehydrogenase, is located at 38.6 min on the map. We have confirmed this location by showing linkage with three Tn10 insertions that are linked to the aroD, pheS, and ansA loci, by complementation by a restriction-mapped lambda clone, and by showing correspondence between the restriction maps of the chromosome and the cloned and sequenced gdhA gene.

Chromosomes, Bacterial↗
PubMed1989

Molecular cloning and restriction endonuclease mapping of two strains of canine adenovirus type 2.

The DNA of a field isolate and of a vaccine strain of canine adenovirus type 2 (CAV-2) were analysed by digestion with several restriction endonucleases. The PstI restriction fragments of the field isolate (CAV-2 Glasgow) and the vaccine strain were cloned into the plasmid pBR322. Physical maps of the two viral genomes were constructed by molecular hybridization of PstI, EcoRI, SmaI, BamHI and KpnI digests of the viral DNA with the cloned PstI fragments. The restriction profile of CAV-2 Glasgow was shown to be virtually identical to those of the two prototype CAV-2 strains, Toronto A26/61 and Manhattan. However, the restriction fragment pattern of the vaccine strain of CAV-2 showed characteristic alterations, in particular additional sequences at or near the genome termini.

Adenoviridae↗
PubMed1993

Construction of a 5.2-megabase physical map of the human X chromosome at Xq22 using pulsed-field gel electrophoresis and yeast artificial chromosomes.

Several genes involved in human genetic diseases map to the Xq22 band on the long arm of the human X chromosome. We have constructed a long-range restriction map of the most proximal part of Xq22. Initially, pulsed-field gel electrophoresis, in combination with rare-cutting restriction enzymes, was used to try and establish physical linkage of 11 polymorphic and nonpolymorphic DNA markers. This approach resulted in the construction of three long-range restriction maps around groups of physically linked Xq22 markers that spanned over 5.0 Mb of DNA. Yeast artificial chromosome clones were used to organize the three long-range maps onto a contiguous 5.2-Mb stretch of Xq22. The order of markers in this region was shown to be cen-GLA-DXS178-DXS101-DXS83-DXS24-DXS101-+ ++DXS54-PLP-DXS94-DXS147-DXS17-DXS87-tel . The results of this study suggest that the proximal part of Xq22 may be rich in genes. Construction of a physical map for this region will, therefore, facilitate the localization and subsequent isolation of novel genes.

Chromosome Mapping↗
PubMed1981

Characterization of cloned rat ribosomal DNA fragments.

Two Charon 4A lambda bacteriophage clones were characterized which contain all and part o the 18S ribosomal DNA of the rat. One clone contained two Eco RI fragments which include the whole 18S ribosomal RNA region and part of 28S ribosomal RNA region. The other clone contained an Eco RI fragment which covers part of 18S ribosomal RNA region. There were differences between the two clones in the non-transcribed spacer regions suggesting that there is heterogeneity in the non-transcribed spacer regions of rat ribosomal genes. The restriction maps of the two clones were compared to the restriction map of the cloned mouse ribosomal DNA. Eco RI, Hind III, Pst I, and Bam HI sites in 18S ribosomal RNA regions were in the same places in mouse and rat DNA but the restriction sites in the 5'-spacer regions were different.

Animals↗
PubMed1978

Mapping of restriction sites in the transforming HpaI-E fragment of adenovirus type 5 DNA.

Adenovirus type 5 (Ad5) DNA was degraded with endo R . HpaI; the left-terminal fragment, HpaI-E has recently been shown to be the smallest segment of Ad5 DNA, that can transform non-permissive cells. This fragment was labelled at its termini by limited exonuclease III digestion followed by repair synthesis with DNA polymerase and alpha-32P-labelled deoxynucleoside triphosphates. It was then further digested with each of the restriction endonucleases HpaII, HaeIII, AluI, HinfI and TaqI; the cleavage products thus obtained were ordered into a physical map.

Adenoviridae↗
PubMed1993

Molecular characterization of a variant Ph1 translocation t(9;22;11) (q34;q11;q13) in chronic myelogenous leukemia (CML) reveals the translocation of the 3'-part of BCR gene to the chromosome band 11q13.

We performed cloning and sequence analysis of translocation junctions at 11q- and 22q- (Ph1) chromosomes and the corresponding germline DNAs of a variant Ph1-positive CML with t(9;22;11)(q34;q11;q13). Southern blot analysis using probes for different regions of bcr mapped the translocation break near the 5'-side of bcr exon 4. Cloning, Southern blot analysis and restriction map analysis of both bcr fragments showed that the part of bcr 3'- to the translocation break moved to 11q13. Sequence analysis of the translocation junction on the Ph1 chromosome showed that the translocation break occurred 63 bp upstream of exon 4. Compared to the germline sequence, bcr sequence from the translocated partners showed deletion of seven basepairs at the site of translocation. A probe derived from the 5'-region of the clone isolated from the 11q- chromosome identified clonal rearrangements in the leukemic DNA. Restriction map and sequence analysis showed that this clone consisted of the 3'-half of the glutathione S-transferase Pi (GST-Pi) gene and the 3'-part of bcr. We identified two point mutations in the GST-Pi allele involved in translocation. Northern blot analysis showed that the GST-Pi gene was expressed in the leukemic cells at blast crisis but not at chronic phase; however, no fusion mRNA between GST-Pi and bcr was identified. We did not find any sequence homology between 11q13 DNA and 22q11 DNA around the translocation breakpoints; however, sequences homologous to ALU repeats were identified close to the sites of translocation breaks at 22q11 and 11q13. This study supports our hypothesis that variant Ph1 translocations may occur as primary cytogenetic changes similar to the classical Ph1 translocations.

Base Sequence↗
PubMed1996

Molecular cloning and restriction enzyme mapping of avian adenovirus type 8 DNA.

Avian adenovirus (AAV) type 8 was cultured in an avian hepatoma cell line designated CH-SAH and the viral DNA extracted and purified. Restriction enzyme analysis of viral DNA using the endonucleases ApaI, EcoRI, HindIII, KpnI, NotI, SpeI, StuI and XbaI was carried out, and fragments representing the entire genome were cloned. According to the restriction enzyme fragments, the size of the AAV type 8 genome was calculated to be 44.7 kb. Subcloning of viral DNA fragments and hybridization studies using selected viral DNA fragments facilitated the construction of the physical map of AAV type 8 DNA.

Animals↗
PubMed1988

Molecular cloning and restriction enzyme mapping of an African swine fever virus isolate from Malawi.

DNA prepared from a field isolate of African swine fever virus, which causes high mortality and severe disease in domestic pigs, was cloned in bacteriophage lambda and plasmid vectors. Clones containing DNA inserts overlapping with each other and together covering the complete genome, apart from short fragments close to the cross-linked termini of the genome, were obtained. A complete restriction enzyme site map of the genome for three enzymes was deduced.

African Swine Fever↗
PubMed1993

The temperate phages RP2 and RP3 of Streptomyces rimosus.

The oxytetracycline-producing Streptomyces rimosus strains R6-65 and R7 (ATCC 10970) are lysogenic for the two narrow-host-range phages RP2 and RP3. Both phages are released at low frequency from the lysogenic strains and form plaques on 'cured' S. rimosus strains. RP2 and RP3 are of similar shape with flexible tails and contain double-stranded DNA of about 70% G+C with cohesive ends (group B1 of bacteriophage classification). The two phages also have identical, very slow, growth kinetics in S. rimosus, with a latent phase of about 6 h and a rise period of about 4 h. RP2 and RP3 are heteroimmune and they differ slightly in their size of phage particles and length of DNA (64.7 and 62.4 kb for RP2 and RP3, respectively). The restriction maps of the two phages are completely different, and hybridization experiments showed only one short region of sequence similarity (less than 430 bp); the two phages are thus essentially unrelated. Both phages lysogenize their hosts by recombination via defined attachment (att) sites. The positions of the attP sites have been localized on the restriction maps of RP2 and RP3 to restriction fragments of 800 and 300 bp, respectively. The prophages did not affect the level of oxytetracycline production or the genetic instability of this trait.

Attachment Sites, Microbiological↗
PubMed1988

Growth-rate-dependent expression and cloning of gnd alleles from natural isolates of Escherichia coli.

6-Phosphogluconate dehydrogenase (6PGD), encoded by gnd, is highly polymorphic among isolates of Escherichia coli form natural populations. As a means of characterizing the growth-rate-dependent regulation of the level of 6PGD, five gnd alleles, including the E. coli B/r allele, were crossed into E. coli K-12 with bacteriophage P1. In each of the isogenic strains, the level of 6PGD was two- to threefold higher in cells grown on glucose than in cells grown on acetate. The level of enzyme activity in the acetate-grown cells varied about sixfold within the set of isogenic strains. The physiological importance of these differences in enzyme level is discussed. The gnd gene was cloned from five E. coli strains and Salmonella typhimurium LT-2 and mapped with twelve restriction endonucleases. gnd was located and oriented on the chromosomal DNAs. The restriction maps of the genes were aligned at conserved restriction sites, and the relative divergence of the genes was estimated from restriction site polymorphisms. The E. coli gnd genes differed from the S. typhimurium gene by about 11%. Most of the E. coli genes differed from one another by less than 5%, but one allele differed from the others by about 10%. Only the gnd gene from E. coli K-12 had an IS5 element located nearby.

Alleles↗
PubMed1990

Geographic variation in human mitochondrial DNA from Papua New Guinea.

High resolution mitochondrial DNA (mtDNA) restriction maps, consisting of an average of 370 sites per mtDNA map, were constructed for 119 people from 25 localities in Papua New Guinea (PNG). Comparison of these PNG restriction maps to published maps from Australian, Caucasian, Asian and African mtDNAs reveals that PNG has the lowest amount of mtDNA variation, and that PNG mtDNA lineages originated from Southeast Asia. The statistical significance of geographic structuring of populations with respect to mtDNA was assessed by comparing observed GST values to a distribution of GST values generated by random resampling of the data. These analyses show that there is significant structuring of mtDNA variation among worldwide populations, between highland and coastal PNG populations, and even between two highland PNG populations located approximately 200 km apart. However, coastal PNG populations are essentially panmictic, despite being spread over several hundred kilometers. Highland PNG populations also have more mtDNA variability and more mtDNA types represented per founding lineage than coastal PNG populations. All of these observations are consistent with a more ancient, restricted origin of highland PNG populations, internal isolation of highland PNG populations from one another and from coastal populations, and more recent and extensive population movements through coastal PNG. An apparent linguistic effect on PNG mtDNA variation disappeared when geography was taken into account. The high resolution technique for examining mtDNA variation, coupled with extensive geographic sampling within a single defined area, leads to an enhanced understanding of the influence of geography on mtDNA variation in human populations.

Australia↗
PubMed1989

Long-range restriction enzyme maps of DNF15S2, D3S2, and c-raf1 loci on the short arm of human chromosome 3.

Physical maps have been constructed around loci DNF15S2, D3S2, and c-raf1 on the short arm of human chromosome 3 using pulsed field gradient gel electrophoresis. The normal restriction pattern has not been altered by a t(3;8)(p14.2;q24.1) characteristic for a hereditary form of renal cell carcinoma, indicating that the breakpoint itself is not included in any of the mapped areas. We have found a CpG island within the DNF15S2 locus, suggesting the presence of a functional gene in the region.

Blotting, Southern↗
PubMed1991

The LCK gene is involved in the t(1;7)(p34;q34) in the T-cell acute lymphoblastic leukemia derived cell line, HSB-2.

HSB-2 is a cell line derived from a patient who had T-cell acute lymphoblastic leukemia (T-cell ALL) with a t(1;7)(p34;q34). We used a genomic probe from the T-cell receptor beta (TCR beta) locus (7q34) to identify DNA rearrangements in HSB-2. Two rearranged BglII DNA fragments were cloned, and one of these clones was shown to contain the translocation breakpoint on the derivative chromosome I [der(I)]. We used a probe derived from this clone to isolate an unrearranged phage clone encompassing the breakpoint at Ip34. The restriction map of this clone was compared to the published maps of known protooncogenes located at Ip32-34. By restriction mapping, Southern blot analysis, and DNA sequencing we showed that the translocation breakpoint on chromosome I is located within the first intron of the LCK gene. The LCK gene codes for p56lck, a member of the SRC family of cytoplasmic tyrosine protein kinases. There are two classes of LCK transcripts (type I and type II), each expressed from a distinct promoter, and each having a unique 5' untranslated region (UTR); the protein coding regions of the two classes are identical. The breakpoint in the t(1;7) separates the two LCK promoters and juxtaposes the constant region of the TCR beta locus with the proximal promoter and with the protein-coding region of the LCK gene on the der(I) chromosome.

Base Sequence↗
PubMed1993

Genomic and yeast artificial chromosome long-range physical maps linking six loci in 10q11.2 and spanning the multiple endocrine neoplasia type 2A (MEN2A) region.

Multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma (FMTC) are dominantly inherited cancers that have in common the clinical feature of medullary thyroid carcinoma (MTC). We have performed both genomic long-range restriction mapping and yeast artificial chromosome (YAC) contig assembly and restriction mapping to establish physical linkage, order, and distances between six loci in 10q11.2 near the genes responsible for these hereditary cancers. RET, D10S94, D10S182, and D10S102 have been mapped in genomic DNA. RET, D10S94, D10S182, D10F38S3, and the 10q11.2 sequences detected by DNA marker DM124 are encompassed by a 1-Mb YAC contig. Six physically linked loci are within 1.4 Mb and have an order and orientation of 10cen, D10F38S3, DM124, RET, D10S94, D10S182, D10S102, 10qter. Mutations in the RET proto-oncogene have recently been demonstrated to be associated with MEN 2A and FMTC. RET is located within a genetically defined MEN2A candidate interval between D10S141 and D10S94; MEN2B has been mapped to a larger, overlapping region between D10S141 and a more distal locus, RBP3. Both our genomic physical map and our YAC contig span the entire MEN2A candidate region and overlap with that of MEN2B. These maps will facilitate the identification of genes that can be considered candidates for MEN2B and the identification of tumor-specific alterations important in sporadic MTC.

Base Sequence↗
PubMed1981

Hind III restriction site map of the human adenovirus type 1 DNA.

The physical map of human adenovirus type 1 DNA was constructed with the Hind III restriction enzyme. Direct comparison of the DNA fragments with those of adenovirus type 2 revealed that the genome of adenovirus type 1 is 200 to 300 base pairs longer. The difference are located outside the region of the inverted terminal repetition within three distinct loci. Fragment Hind III-F of type 1 DNA which is presumed to carry all the genes responsible for in vitro transformation can be separated without considerable contamination by a single electrophoretic step.

Adenoviruses, Human↗
PubMed1977

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.

DNA Restriction Enzymes↗