Adenylate kinase isoenzymes in Aspergillus nidulans

Multiple HindIII-restriction fragments of Salmonella typhimurium and Salmonella typhi chromosomal DNA exhibited homology with the heat-labile enterotoxin (LT1) gene of Escherichia coli as determined by Southern blot analysis. A 9.4 kb HindIII restriction fragment identified in S. typhimurium and S. typhi chromosomal DNA reacted with both eltA and eltB gene probes. However, the homology of the 9.4 kb DNA fragment from these Salmonella species was greater with eltB than eltA. In addition, a synthetic oligonucleotide probe, made to a portion of the putative GM1-ganglioside binding region of cholera toxin (CT) and LT1, hybridized with the 9.4 kb DNA fragment of S. typhimurium but not with the 9.4 kb fragment found in S. typhi isolates. The hybridization of multiple restriction fragments of Salmonella DNA with eltA and eltB gene sequences further suggests duplication of the stx operon on the chromosome of these bacteria.     

Size and physical map of the chromosome of Haemophilus influenzae.

A variation of pulse-field electrophoresis, field-inversion gel electrophoresis, was used to determine the size and physical map of the chromosome of Haemophilus influenzae. The DNA of H. influenzae had a low G + C content (39%) and no restriction sites for the enzymes NotI or SfiI. However, a number of restriction enzymes (SmaI, ApaI, NaeI, and SacII) that recognized 6-base-pair sequences containing only G and C nucleotides were found to generate a reasonable number of DNA fragments that were separable in agarose gels by field-inversion gel electrophoresis. The sizes of the DNA fragments were calibrated with a lambda DNA ladder and lambda DNA restriction fragments. The sum of fragment sizes obtained with restriction digests yielded a value for the chromosome of 1,980 kilobase pairs. Hybridization of a labeled fragment with two or more fragments from a digest with a different restriction enzyme provided the information needed to construct a circular map of the H. influenzae chromosome.     

Properties of a membrane-attached form of the folded chromosome of Escherichia coli

Two types of DNA-containing particles are released from lysozyme-produced Escherichia coli spheroplasts after gentle lysis with non-ionic detergents in 1.-0 m-NaCl. Lysis at 25 °C releases the folded chromosomes (1300 S to 2200 S particles). Lysis at 10 °C results in faster sedimenting structures (3000 S to 4000 S). Both types of particles coexist in extracts of cells lysed at intermediate temperatures, i.e. 15 °C.The 3000 S to 4000 S particles are folded chromosomes attached to membrane fragments; they contain membrane proteins and phospholipids in addition to the folded DNA and nascent RNA chains. Incubation of the membrane-attached chromosomes with 1% Sarkosyl releases the folded chromosomes; this Sarkosyl treatment removes the membrane proteins and phospholipids, and halves the sedimentation velocity of the particles, but has no effect on the folded DNA and nascent RNA chains.Membrane-attached chromosomes cannot be isolated from amino acid-starved cells which have completed their rounds of DNA replication; all of the DNA then appears as released folded chromosomes. After resumption of protein synthesis, chromosome attachment to the membrane precedes the initiation of DNA replication. Controls strongly suggest that the changes observed, i.e. the attachment and release from the membrane of the folded chromosome, are related to the act of DNA replication itself.     

Random-breakage mapping method applied to human DNA sequences.

The random-breakage mapping method [Game et al. (1990) Nucleic Acids Res., 18, 4453-4461] was applied to DNA sequences in human fibroblasts. The methodology involves NotI restriction endonuclease digestion of DNA from irradiated calls, followed by pulsed-field gel electrophoresis, Southern blotting and hybridization with DNA probes recognizing the single copy sequences of interest. The Southern blots show a band for the unbroken restriction fragments and a smear below this band due to radiation induced random breaks. This smear pattern contains two discontinuities in intensity at positions that correspond to the distance of the hybridization site to each end of the restriction fragment. By analyzing the positions of those discontinuities we confirmed the previously mapped position of the probe DXS1327 within a NotI fragment on the X chromosome, thus demonstrating the validity of the technique. We were also able to position the probes D21S1 and D21S15 with respect to the ends of their corresponding NotI fragments on chromosome 21. A third chromosome 21 probe, D21S11, has previously been reported to be close to D21S1, although an uncertainty about a second possible location existed. Since both probes D21S1 and D21S11 hybridized to a single NotI fragment and yielded a similar smear pattern, this uncertainty is removed by the random-breakage mapping method.     

Characterization of a cloned ribosomal fragment from mouse which contains the 18S coding region and adjacent spacer sequences.

The large EcoRI fragment of mouse ribosomal genes containing parts of the non-transcribed spacer, the external transcribed spacer located at the 5' end of the precursor molecule and about two thirds of the 18S sequence has been cloned in bacteriophage lambda gtWES. A physical map of the DNA was constructed by cleavage with several restriction endonucleases and hybridization of the restriction fragments of the recombinant DNA with labelled 18S and 45S rRNA. The orientation of the inserted fragment as well as the length of the 18S sequence was determined by electron microscopy of R-loop containing molecules. The absence of hybridization of the cloned fragment to other fragments in the genome shows that the non-transcribed spacer does not have a significant length of sequences in common with other sequences in the genome.     

Association of the folded chromosome with the cell envelope of E. coli: Characterization of the proteins at the DNA-membrane attachment site

Gentle lysis of E. coli cells in the presence of a DNA counterion (either 1.0 M NaCl or 5 mM spermidine) permits the isolation of the folded intact bacterial chromosome associated with membrane fragments. Most of the proteins in these chromosomes are also found in purified membrane preparations, and they can be identified as belonging to either the inner or the outer bacterial membrane.Ultraviolet irradiation of the membrane-attached chromosomes causes the formation of a stable complex between two inner membrane proteins (molecular weight 80,000 and 56,000 daltons) and 5-bromodeoxyuridine (BrdU)-substituted DNA. The photochemical attachment of BrdU-substituted DNA to specific membrane proteins suggests that these proteins may be bound to the DNA in vivo. Such DNA-membrane-binding proteins may have a role in the attachment of the folded chromosome to the bacterial envelope.     

Cloning and template activity of the origins of replication of phage phi 29 DNA

A 73-bp fragment from the left end of phi 29 DNA and a 269-bp fragment from the right end have been cloned in plasmids pPLc28 and pKK223-3, respectively, after removal of the terminal protein p3 by treatment with piperidine. In addition, the 73- and 269-bp fragments were cloned together in plasmid pKK223-3 in such a way that the two termini of phi 29 DNA were joined. Treatment of the latter recombinant plasmid with AhaIII releases several fragments, two of which contain the phi 29 DNA terminal sequences at the DNA end. These two fragments initiated replication specifically at the ends of the DNA giving rise to the formation of the p3-dAMP complex. The activity was about 15% of that obtained with phi 29 DNA-protein p3. All remaining recombinant plasmids were essentially inactive when tested as templates either in circular form or after cutting in such a way that placed the origin of phi 29 DNA replication close but not at the DNA end.     

Human DNA sequences isolated with an immunoglobulin switch region probe: sequence,chromosomal localization,and restriction fragment length polymorphisms

Summary We have screened a human genomic DNA library with an immunoglobulin (Ig) derived switch (S) region specific probe for homologous sequences. Five Ig independent phage clones were isolated and characterized. The S sequence homologous DNA fragments are short compared to the S region sequences. Ig independent S sequences are flanked by highly repetitive DNA elements and perfect inverted repeats can be demonstrated in their close vicinity. Using subclones of S homologous sequences restriction fragment length polymorphisms were shown within DNA of different T cell leukemias. Burkitt lyphhomas, lymphoblastoid cell lines, and DNA of healthy individuals. One of the five clones isolated with the S region probe was evidently localized to chromosome 2 and/or 40 and showed a complex hybridisation pattern with several different human DNAs. S homologous sequences of another clone are most likely localized on chromosome 1. It is possible that these Ig indenpendent S sequences have arisen by amplification and transposition and that they are involved in genetic recombination.     

Identification and classification of S haplotypes in Raphanus sativus by PCR-RFLP of the S locus glycoprotein (SLG) gene and the S locus receptor kinase (SRK) gene

Polymorphism of the S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes in Raphanus sativus was analyzed by PCR-RFLP using SLG- and SRK-specific primers. Twenty four inbred lines of R. sativus could be grouped into nine S haplotypes. DNA fragments of SLG alleles specifically amplified from five S haplotypes by PCR with Class-I SLG-specific primers showed different profiles upon polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. The five R. sativus SLG alleles were determined for their nucleotide sequences of DNA fragments. Comparison of the amino-acid sequences with a reported Brassica SLG (S₆) showed 77-84% homology. Deduced amino-acid sequences showed 12-conserved cystein residues and three hypervariable regions which are characteristic of Brassicsa SLG. A DNA fragment was also amplified by PCR from two of each S haplotype with Class-II SLG-specific primers, and showed polymorphism when cleaved with restriction endonucleases. The nucleotide sequences of amplified DNA fragments of the Class-II SLG revealed about 60% similarity with those of the Class-I SLG. It is concluded that there exist both Class I and Class II S alleles in R. sativus, as in Brassica campestris and Brassica oleracea. PCR using SRK-specific primers amplified a DNA fragment of about 1.0 kb from seven of each S haplotype out of 24 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. Nucleotide sequences of the DNA fragments amplified from the seven S haplotypes showed that the fourth and the fifth exons of SRK are highly conserved, and that there is high variation in the fifth intron, the sixth intron and seventh exon of the SRK which may be responsible for the polymorphic band patterns in PCR-RFLP analysis. The PCR-RFLP method has proven useful for the identification of S alleles in inbred lines and for listing S haplotypes in R. sativus. Phylogenic analysis of the SLG and SRK sequences from Raphanus and Brassica revealed that the Raphanus SLGs and SRKs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs and SRKs. Furthermore, SLGs and SRKs from Raphanus were both grouped into Class-I or Class-II S haplotypes. Therefore, these results suggest that the diversification of the SLG and SRK alleles occurred prior to the differentiation of the two genera Brassica and Raphanus.     

An extremely polymorphic locus on the short arm of the human X chromosome with homology to the long arm of the Y chromosome.

A genomic DNA clone named CRI-S232 reveals an array of highly polymorphic restriction fragments on the X chromosome as well as a set of non-polymorphic fragments on the Y chromosome. Every individual has multiple bands, highly variable in length, in every restriction enzyme digest tested. One set of bands is found in all males, and co-segregates with the Y chromosome in families. These sequences have been regionally localized by deletion mapping to the long arm of the Y chromosome. Segregation analysis in families shows that all of the remaining fragments co-segregate as a single locus on the X chromosome, each haplotype consisting of three or more polymorphic fragments. This locus (designated DXS278) is linked to several markers on Xp, the closest being dic56 (DXS143) at a distance of 2 cM. Although it is outside the pseudoautosomal region, the S232 X chromosome locus shows linkage to pseudoautosomal markers in female meiosis. In determining the X chromosome S232 haplotypes of 138 offspring among 19 families, we observed three non-parental haplotypes. Two were recombinant haplotypes, consistent with a cross-over among the S232-hybridizing fragments in maternal meiosis. The third was a mutant haplotype arising on a paternal X chromosome. The locus identified by CRI-S232 may therefore be a recombination and mutation hotspot.     

Specific cloning of DNA fragments unique to the dog Y chromosome

A novel technique that enabled the specific cloning of a DNA fragment unique to the dog Y chromosome is described. The method involves competitive hybridization of DNA prepared from male dog lymphocytes with biotin-labeled DNA prepared from female dog lymphocytes. The biotinylated female-female and male-female hybrid DNA fragments were removed by capture with streptavidin-coated paramagnetic particles. Full-length double-stranded DNA was generated from the remaining fragments by using the Klenow fragment of DNA polymerase I, followed by direct cloning using a low-background ligation technique. Analysis of putative recombinant clones derived by this method has led to the identification of a fragment that hybridizes specifically to male dog DNA. The clones were selected initially on the basis of a differential signal obtained when hybridized to dilutions of male and female dog DNA immobilized on neutral nylon membrane. To evaluate its suitability as a probe for trans-sexually grafted cells in transplantation studies, the fragment was labeled with digoxigenin and hybridized in situ to male and female dog tissue sections. The clone designated number 6.2 hybridized strongly to male dog nuclei. The cloning strategy employed could be extended to other studies in which competitive reassociation can be used to identify unique DNA sequences.     

The cyclization of Xenopus laevis 5 S DNA

DNA from Xenopus laevis containing the sequences complementary to 5 S RNA has been studied by the formation of folded rings. Maximal cyclization for fragments 1 to 2 μm in length is 45 to 55%. Thus the efficiency of folded ring formation from this tandemly-repeating DNA is about 50%, assuming that all fragments are 5 S DNA. From the ring frequency as a function of the number of nucleotides removed from the 3′ terminals of the shear-broken fragments, one may calculate that the repeating sequence is approximately 750 nucleotides long, a number that agrees with earlier partial denaturation mapping. The circumference of the folded rings confirms this repeating length since most rings correspond to modular size classes of 0.25-μm increments. Fragments 12 μm long cyclize almost as readily as 1 to 2-μm fragments do. Therefore, the length of the regions (g-regions) containing the tandemly-repeating 5 S DNA is more than 12 μm. The folded rings are about as stable to linearization by increasing concentrations of formamide as the duplex DNA is to denaturation. This indicates that the local, non-transcribed, spacer portions which represent the majority (83%) of the nucleotides in the tandemly-repeating unit, are probably homogeneous in sequence. The exonuclease-treated 5 S DNA fragments cyclize more rapidly than phage T7 DNA, and the kinetics are in accord with theoretical expectation.     

Cloning the complete human adenine phosphoribosyl transferase gene

We have isolated a clone from a human genomic lambda library which cross-hybridises with the cloned hamster adenine phosphoribosyl transferase gene (aprt). After restriction mapping and further hybridisation to the hamster gene, a series of putative human aprt-containing fragments has been isolated and tested for ability to transform adenine phosphoribosyl transferase-deficient (aprt-) strains of Chinese hamster ovary (CHO) cells to APRT proficiency. Transforming activity was detected in a 48-kb lambda clone, the 17.4-kb EcoRI insert, and an 8.6-kb HincII fragment. Smaller fragments have thus far shown no transforming activity. Transformants appear to be stable for the APRT+ phenotype, and human aprt DNA sequences are present in the hamster transformants. The 8.6-kb HincII fragment has been subcloned and the insert mapped. Nonrepetitive regions of this subclone have been identified, and should prove valuable for chromosome walking studies on human chromosome 16, familial studies of a human aprt- trait, the analysis of restriction fragment length polymorphisms (RFLPs) in the area surrounding the aprt gene, and the fine structure mapping of the mutations induced by chemical carcinogens and alkylating agents.     

Localization of the active gene of aldolase on chromosome 16, and two aldolase A pseudogenes on chromosomes 3 and 10

Summary Southern blot analysis of human genomic DNA hybridized with a coding region aldolase A cDNA probe (600 bases) revealed four restriction fragments with EcoRI restriction enzyme: 7.8 kb, 13 kb, 17 kb and >30 kb. By human-hamster hybrid analysis (Southern technique) the principal fragments, 7.8 kb, 13 kb, >30 kb, were localized to chromosomes 10, 16 and 3 respectively. The 17-kb fragment was very weak in intensity; it co-segregated with the >30-kb fragment and is probably localized on chromosome 3 with the >30-kb fragment. Analysis of a second aldolase A labelled probe protected against S1 nuclease digestion by RNAs from different hybrid cells, indicated the presence of aldolase A mRNAs in hybrid cells containing only chromosome 16. Under the stringency conditions used, the EcoRI sequences detected by the coding region aldolase A cDNA probe did not correspond to aldolase B or C. The 7.8-kb and >30-kb EcoRI sequences, localized respectively on chromosomes 10 and 3, correspond to aldolase A pseudogenes, the 13-kb EcoRI sequence localized on chromosome 16 corresponds to the aldolase active gene. The fact that the aldolase A gene and pseudogenes are located on three different chromosomes supports the hypothesis that the pseudogenes originated from aldolase A mRNAs, copied into DNA and integrated in unrelated chromosomal loci.     

DNA profiling by detection of repetitive nucleotide sequences on human chromosome 6

Genetic/genomic polymorphism, i.e. variations in DNA sequences are ideally assayed by direct nucleotide sequencing of a gene region or other homologous segment of the genome. An easier and cheaper approach, however, if the variants are analyzed by hybridization technology using restriction fragment length polymorphisms (RFLPs) or by detection of the number of tandem repeats (VNTR) of small DNA segments, the "minisatellites". In this study we describe results of the DNA analysis of repetitive sequences of human 6th chromosome by the application of a chemiluminescent labeled probes. The allele frequency distribution of polymorphic DNA sequences has been determined in unrelated individuals. The isolated genomic DNA was cut with Pst I restriction enzyme, size fractionated on agarose gel and hybridized with a chemiluminescent labeled D6 S132 probe. At this locus the Pst I cleaved DNA fragments are ranging from 1841 to 6098 base pairs (bp). Specific genetic pattern was characterized by more frequent fragments (3313 and 3884 bp), and the rarely occurring ones (clustered between 1841-2595 and 5227-6098 bp). Our study provides a further possibility for characterization of individual genomic patterns.     

Isolation and characterization of two repetitive DNA fragments located near the centromere of the mouse X chromosome

Two repetitive DNA fragments located on the mouse X chromosome are described. The fragments were isolated from a lambda phage library enriched in X-chromosomal sequences by flow sorting. Both fragments, which are repeated 20 to 50 times in the genome, were mapped to the mouse X chromosome by Southern blot hybridization to DNA from hybrid cells retaining the mouse X chromosome, by dosage analysis, and by in situ hybridization to mouse chromosomes. In mouse strain C57BL/10BK, one fragment appeared to be located only on the X chromosome, while the other fragment had homologous sequences on chromosome 11 in addition to the X chromosome. The latter fragment showed DNA variants between mouse strains, which are potentially useful for mapping. Both fragments cross-hybridized to another mouse species: Mus caroli. In this species, each fragment appeared to be located on the X chromosome, indicating that some X-chromosome repetitive sequences are partially conserved. In addition, one fragment cross-hybridized to human DNA.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.