DNA loop domains in a 1.4-Mb region around the human hprt gene mapped by cleavage mediated by nuclear matrix-associated topoisomerase II

We have mapped the positions in a ～1.4-Mb region of genomic DNA around the human hprt gene which are accessible in vivo to cleavage by topoisomerase II associated with the nuclear matrix. These positions, which are interpreted as the boundaries of DNA loop domains, were mapped in K562 cells by examining the truncation of rare-cutter restriction fragments separated by pulsed field gel electrophoresis after topoisomerase II-mediated cleavage, using seven linked markers mapped in this region as probes for indirect end-labeling. Eleven cleavage positions were detected and were interpreted as defining ten loop domains of lengths between 70 and 210?kb (average ～135?kb); the hprt gene resides in a 150-kb loop domain. Loop domain boundaries coincided with three of the fifteen deletion breakpoints mapped in a 600-kb sector of this region in human lymphocytes, within the limits of resolution of pulsed field gel electrophoresis; this correlation was not statistically significant.     

Genetic and physical analysis of a YAC contig spanning the fungal disease resistance locus Asc of tomato (Lycopersicon esculentum)

The Alternaria stem canker disease of tomato is caused by the necrotrophic fungal pathogen Alternaria alternata f. sp. lycopersici (AAL). The fungus produces AAL toxins that kill the plant tissue. Resistance to the fungus segregates as a single locus, called Asc, and has been genetically mapped on chromosome 3 of tomato. We describe here the establishment of a 1383-kb YAC contig covering the Asc locus and a series of plants selected for recombination events around the Asc locus. It was shown that the YAC contig corresponds to a genetic distance of at least 11.2 cM. Thus, the recombination rate in the Asc region is six times higher (123 kb/cM) than the average for the tomato genome. Furthermore, the Asc locus could be localised to a 91-kb fragment, thus paving the way for the cloning and identification of the Asc gene(s) by complementation. Received: 31 July 1998 / Accepted: 6 October 1998     

Molecular cloning, characterization, and chromosomal localization of the mouse homologue of CD84, a member of the CD2 family of cell surface molecules

 CD84 is a member of the immunoglobulin gene superfamily (IgSF) with two Ig-like domains expressed primarily on B lymphocytes and macrophages. Here we describe the cloning of the mouse homologue of human CD84. Mouse CD84 cDNA clones were isolated from a macrophage library. The nucleotide sequence of mouse CD84 was shown to include an open reading frame encoding a putative 329 amino acid protein composed of a 21 amino acid leader peptide, two extracellular immunoglobulin (Ig)-like domains, a hydrophobic transmembrane region, and an 87 amino acid cytoplasmic domain. Mouse CD84 shares 57.3% amino acid sequence identity (88.7%, considering conservative amino acid substitutions) with the human homologue. Chromosome localization studies mapped the mouse CD84 gene to distal chromosome 1 adjacent to the gene for Ly-9, placing it close to the region where other members of the CD2 IgSF (CD48 and 2B4) have been mapped. Northern blot analysis revealed that the expression of mouse CD84 was predominantly restricted to hematopoietic tissues. Two species of mRNA of 3.6 kilobases (kb) and 1.5 kb were observed. The finding that the pattern of expression was restricted to the hematopoietic system and the conserved sequence of the mouse CD84 homologue suggests that the function of the CD84 glycoprotein may be similar in humans and mice. Received: 1 July 1998 / Revised: 31 August 1998     

Refined genetic map of the obligate methylotroph Methylobacillus flagellatum

We present a refined genetic map of the obligate methylotroph Methylobacillus flagellatum. New, Hfr (high-frequency-of-transfer) donors, and pulsed-field gel electrophoresis, were used to determine that M.␣flagellatum contains one ∼3.1-Mb circular chromosome, and no plasmids. A correlation between time-of-entry units and DNA length was established. Using in vivo and in vitro cloning, and sequencing, a number of new genetic markers were identified and mapped; in addition, the nature of some of the previously mapped markers was elucidated. Received: 11 August 1997 / Accepted: 11 December 1997     

Mapping of genomic DNA loop organization in a 500-kilobase region of the Drosophila X chromosome by the topoisomerase II-mediated DNA loop excision protocol.

The recently developed procedure of chromosomal DNA loop excision by topoisomerase II-mediated DNA cleavage at matrix attachment sites (S. V. Razin, R. Hancock, O. Iarovaia, O. Westergaard, I. Gromova, and G. P. Georgiev, Cold Spring Harbor Symp. Quant. Biol. 58:25-35, 1993; I. I. Gromova, B. Thompsen, and S. V. Razin, Proc. Natl. Acad. Sci. USA 92:102-106, 1995) has been employed for mapping the DNA loop anchorage sites in a 500-kb region of the Drosophila melanogaster X chromosome. Eleven anchorage sites delimiting 10 DNA loops ranging in size from 20 to 90 kb were found within this region. Ten of these 11 anchorage sites colocalize with previously mapped scaffold attachment regions. However, a number of other scaffold attachment regions are found to be located in loop DNA.     

Mapping of a human centromere onto the DNA by topoisomerase II cleavage

We have mapped the positions of topoisomerase II binding sites at the centromere of the human Y chromosome using etoposide-mediated DNA cleavage. A single region of cleavage is seen at normal centromeres, spanning ~50 kb within the centromeric alphoid array, but this pattern is abolished at two inactive centromeres. It therefore provides a marker for the position of the active centromere. Although the underlying centromeric DNA structure is variable, the position of the centromere measured in this way is fixed relative to the Yp edge of the array, and has retained the same position for >100 000 years.     

DNA loop organization and DNA fragmentation during radiation-induced apoptosis in human lymphocytes

Apoptotic DNA fragmentation induced by gamma-rays has been compared with the DNA loop sizes in G0-human lymphocytes using pulsed field gel electrophoresis (PFGE). Genomic DNA was cleaved into the DNA loops at the topoisomerase II mediated attachment points using short treatment of cells with etoposide. The apoptotic fragmentation, with a distinct cut-off around 50 kb for a maximum length of fragments, appeared 5 h after irradiation when the most part of radiation-induced DNA double strand breaks (DSBs) have been repaired. The data indicate that apoptotic fragmentation of DNA in the G0-human lymphocytes begins when repair of radiation-induced DSBs has been completed. Similar apoptotic DNA fragmentation was also observed following the treatment of cells with etoposide. All genomic DNA was fragmented into 50-kb fragments during the final stages of apoptosis. Most of the DNA in resting lymphocytes is organized into Mb-size loops but loops of sizes down to 50 kb were also observed. A sharp border between the size distributions of DNA loops and apoptotic fragments was found. The data suggest that 50 kb apoptotic fragmentation is not based on excision of the DNA loops. No apoptotic fragments with the sizes more than 5.7 Mb were seen during the whole course of apoptosis. This observation indicates that despite intensive apoptotic fragmentation into the 50-kb fragments the chromosomes maintain integrity during radiation-induced apoptosis in human lymphocytes. We propose a model for radiation-induced apoptotic fragmentation in human lymphocytes that involves four stages: induction of DNA breaks and relaxation of DNA loops; DNA repair followed by reorganization of the DNA loops into the 50-kb units of condensed chromatin; co-operative fragmentation of the reorganized DNA loops into the distinct 50-kb fragments and resealing of the chromosome ends at the sites of this fragmentation; cleavage of the 50-kb fragments at the internucleosomal spacers.     

DNA loop anchorage region colocalizes with the replication origin located downstream to the human gene encoding lamin B2

The recently developed procedure of topoisomerase II-mediated DNA loop excision has been used to analyze the topological organization of a human genome fragment containing the gene encoding lamin B2 and the ppv1 gene. A 3.5 kb long DNA loop anchorage/topoisomerase II cleavage region was found within the area under study. This region includes the end of the lamin B2 coding unit and an intergenic region where an origin of DNA replication was previously found. These observations further corroborate the hypothesis that DNA replication origins are located at or close to DNA loop anchorage regions. J. Cell. Biochem. 69:13–18, 1998. © 1998 Wiley-Liss, Inc.     

Higher order chromatin structures in maize and Arabidopsis.

We are investigating the nature of plant genome domain organization by using DNase I- and topoisomerase II-mediated cleavage to produce domains reflecting higher order chromatin structures. Limited digestion of nuclei with DNase I results in the conversion of the >800 kb genomic DNA to an accumulation of fragments that represents a collection of individual domains of the genome created by preferential cleavage at super-hypersensitive regions. The median size of these fragments is approximately 45 kb in maize and approximately 25 kb in Arabidopsis. Hybridization analyses with specific gene probes revealed that individual genes occupy discrete domains within the distribution created by DNase I. The maize alcohol dehydrogenase Adh1 gene occupies a domain of 90 kb, and the maize general regulatory factor GRF1 gene occupies a domain of 100 kb in length. Arabidopsis Adh was found within two distinct domains of 8.3 and 6.1 kb, whereas an Arabidopsis GRF gene occupies a single domain of 27 kb. The domains created by topoisomerase II-mediated cleavage are identical in size to those created by DNase I. These results imply that the genome is not packaged by means of a random gathering of the genome into domains of indiscriminate length but rather that the genome is gathered into specific domains and that a gene consistently occupies a discrete physical section of the genome. Our proposed model is that these large organizational domains represent the fundamental structural loop domains created by attachment of chromatin to the nuclear matrix at loop basements. These loop domains may be distinct from the domains created by the matrix attachment regions that typically flank smaller, often functionally distinct sections of the genome.     

Patterns of meiotic double-strand breakage on native and artificial yeast chromosomes

The preferred positions for meiotic double-strand breakage were mapped on Saccharomyces cerevisiae chromosomes I and VI, and on a number of yeast artificial chromosomes carrying human DNA inserts. Each chromosome had strong and weak double-strand break (DSB) sites. On average one DSB-prone region was detected by pulsed-field gel electrophoresis per 25 kb of DNA, but each chromosome had a unique distribution of DSB sites. There were no preferred meiotic DSB sites near the telomeres. DSB-prone regions were associated with all of the known ”hot spots” for meiotic recombination on chromosomes I, III and VI. Received: 19 March 1996; in revised form: 26 July 1996 / Accepted: 18 August 1996     

Localization of the sapA gene on a physical map of Campylobacter fetus chromosomal DNA

     We constructed a physical map of Campylobacter fetus TK(+) chromosomal DNA digested by either SmaI, SalI, or NotI using pulsed-field gel electrophoresis and Southern hybridization data. The genome size of C. fetus TK(+) is 2016 kb, larger than that reported by the others. To locate the sapA gene, which encodes the surface array protein (SAP), on the physical map, we performed Southern hybridizations with probes based on the conserved region of the sapA gene. The results showed that more than seven copies of the conserved region were present on C. fetus chromosomal DNA and that the sapA gene was located on a limited number of fragments forming a cluster of genes. By comparing fingerprint patterns of strain TK(+) and strain TK(–), which lost the ability to produce SAP during culture on agar medium, an approximately 10 kb deletion was observed in the fragments of strain TK(–). The results of Southern hybridization with two probes, one from the upstream region and the other from the variable region of sapA, suggest that the loss of SAP expression might not be the result of the loss of the sapA gene itself, but only a loss of its control systems. Received: 25 May 1994 / Accepted: 1 September 1994     

New chromosome 21 DNA markers isolated by pulsed field gel electrophoresis from an ETS2-containing Down syndrome chromosomal region

To generate new chromosome 21 markers in a region that is critical for the pathogenesis of Down syndrome (D21S55-MX1), we used pulsed field gel electrophoresis (PFGE) to isolate a 600-kb NruI DNA fragment from the WA17 hybrid cell line, which has retained chromosome 21 as the only human material. This fragment, which contains the oncogene ETS2, was used to construct a partial genomic library. Among the 14 unique sequences that were isolated, 3 were polymorphic markers and contained sequences that are conserved in mammals. Five of these markers mapped on the ETS2-containing NruI fragment and allowed us to define an 800-kb high-resolution PFGE map.     

Macrorestriction Analysis of Caenorhabditis Elegans Genomic DNA

The usefulness of genomic physical maps is greatly enhanced by linkage of the physical map with the genetic map. We describe a ``macrorestriction mapping' procedure for Caenorhabditis elegans that we have applied to this endeavor. High molecular weight, genomic DNA is digested with infrequently cutting restriction enzymes and size-fractionated by pulsed field gel electrophoresis. Southern blots of the gels are probed with clones from the C. elegans physical map. This procedure allows the construction of restriction maps covering several hundred kilobases and the detection of polymorphic restriction fragments using probes that map several hundred kilobases away. We describe several applications of this technique. (1) We determined that the amount of DNA in a previously uncloned region is <220 kb. (2) We mapped the mes-1 gene to a cosmid, by detecting polymorphic restriction fragments associated with a deletion allele of the gene. The 25-kb deletion was initially detected using as a probe sequences located ~400 kb away from the gene. (3) We mapped the molecular endpoint of the deficiency hDf6, and determined that three spontaneously derived duplications in the unc-38-dpy-5 region have very complex molecular structures, containing internal rearrangements and deletions.     

Cloning and characterization of the gene encoding a repressible acid phosphatase (PHO1) from the methylotrophic yeast Hansenula polymorpha

A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M _r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression. Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998     

Identification of 1-aminocyclopropane-1-carboxylic acid synthase genes controlling the ethylene level of ripening fruit in Japanese pear (Pyrus pyrifolia Nakai)

The shelf life of Japanese pear fruit is determined by its level of ethylene production. Relatively high levels of ethylene reduce storage potential and fruit quality. We have identified RFLP markers tightly linked to the locus that determines the rate of ethylene evolution in ripening fruit of the Japanese pear. The study was carried out using sequences of two types of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (PPACS1 and pPPACS2) and a ACC oxidase gene (PPAOX1) as probes on 35 Japanese pear cultivars expressing different levels of ethylene (0.0∼300 μl/kg fresh weight/h) in ripening fruit. When total DNA was digested with HindIII and probed with pPPACS1, we identified a band of 2.8 kb which was specific to cultivars having very high ethylene levels (≧10 μ1/kg f.w./h) during fruit ripening. The probe pPPACS2 identified a band of 0.8 kb specific to cultivars with moderate ethylene levels (0.5 μl/kg f.w./h–10 μl/kg f.w./h) during fruit ripening. The cultivars that produce high levels of ethylene possess at least one additional copy of pPPACS1 and those producing moderate levels of ethylene have at least one additional copy of pPPACS2. These results suggest that RFLP analysis with different ACC synthase genes could be useful for predicting the maximum ethylene level during fruit ripening in Japanese pear. Received: 1 July 1998 / Accepted: 6 October 1998     

Properties of a cold-active protease from psychrotrophic Flavobacterium balustinum P104

Protease activity was detected in the culture medium of Flavobacterium balustinum P104 grown at 10 °C, which was isolated from salmon (Oncorhynchus keta) intestine. The enzyme, designated as CP-70 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographyies. The molecular mass of the protease was 70 kDa, and its isoelectric point was close to 3.5. Maximal activity toward azocasein was observed at 40 °C and from pH 7.0 to 9.0. The activity was strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that the enzyme is a serine protease. The n-terminal amino acid sequence was Asp-Thr-Arg-Gln-Leu-Leu-Asn-Ala-Asn-Ser-Asp-Leu-Leu-Asn-Thr-Thr-Gly-Asn-Val-Thr-Gly-Leu-Thr-Gly-Ala-Phe-Asn-Gly-Glu-Asn. A search through the database for sequence homology yielded no significant match. The initial cleavage sites for oxidized insulin B-chain were found to be the Glu13-Ala14 and Phe24-Phe25 bonds. The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-70 protease has a broader specificity than the other cold-active proteases against the peptide substrate. Received: 17 April 1998 / Received last revision: 17 June 1998 / Accepted: 10 July 1998     

Gene arrangement of the killer cell inhibitory receptor family on human chromosome 19q13.4 detected by fiber-FISH

 Human chromosome 19q13.4 has recently been revealed to be a remarkable region harboring multiple receptor genes of the immunoglobulin (Ig) superfamily differentially expressed on hematopoietic cell lineages. Over the past few years, more than 50 cDNAs have been cloned for the natural killer cell inhibitory receptor (KIR) gene family, which possess two or three Ig-like domains in the extracellular region. In this study, using two genomic DNA probes containing intron sequences of genes corresponding to the two- and three-domain types, we applied two-color-fluorescence in situ hybridization on stretched DNA fiber preparations (fiber-FISH). As a result, 11 positions homologous to KIR genes were found as a cluster within a range of approximately 120 kilobases on a chromatin fiber from human chromosome 19. Received: 7 January 1998 / Revised: 25 February 1998     

<Emphasis Type="Italic">Arabidopsis</Emphasis> sister chromatids often show complete alignment or separation along a 1.2-Mb euchromatic region but no cohesion “hot spots”

Sister chromatid cohesion is a prerequisite for correct segregation and possibly other functions of replicated chromosomes. Except for yeast, no details are known about arrangement of cohesion sites along interphase chromosomes. Within nuclei of several higher plants, sister chromatids are frequently not aligned at various positions along chromosome arms. Therefore, we tested whether preferential alignment positions (“cohesion hot spots”) and constant extension of and distances between aligned sites occur in plants. Along a ~1.2-Mb contig from the bottom arm of chromosome 1, the sister chromatid positions of 13 individual BAC inserts were found to be aligned for ~67–77% of homologues in 4C Arabidopsis thaliana nuclei. The differences between the 13 BAC positions were not significant at the P < 0.01 level. This suggests variability of alignment positions between cells and indicates the absence of cohesion “hot spots”. Similar as for single BACs, FISH with the entire contig indicated complete alignment for ~69% and complete separation of sister chromatids for ~31% of homologues in 4C nuclei. Partial alignment or separation was barely detectable. When three BAC inserts from a 760-kb region were tested simultaneously, alignment or separation of only the central BAC occurred in 3.3% and 3.5% of replicated chromosomes, respectively. Thus, we assume that sister chromatids can be separated or aligned within a Mb range in differentiated cells. However, the minimum extension of aligned sites or distances between them may (in rare cases) fall below ~500 kb.