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PubMed1994

Molecular variation in chloroplast DNA regions in ancestral species of wheat.

Restriction map variation in two 5-6-kb chloroplast DNA regions of five diploid Aegilops species in the section Sitopsis and two wild tetraploid wheats, Triticum dicoccoides and Triticum araraticum, was investigated with a battery of four-cutter restriction enzymes. A single accession each of Triticum durum, Triticum timopheevi and Triticum aestivum was included as a reference. More than 250 restriction sites were scored, of which only seven sites were found polymorphic in Aegilops speltoides. No restriction site polymorphisms were detected in all of the other diploid and tetraploid species. In addition, six insertion/deletion polymorphisms were detected, but they were mostly unique or species-specific. Estimated nucleotide diversity was 0.0006 for A. speltoides, and 0.0000 for all the other investigated species. In A. speltoides, none of Tajima's D values was significant, indicating no clear deviation from the neutrality of molecular polymorphisms. Significant non-random association was detected for three combinations out of 10 possible pairs between polymorphic restriction sites in A. speltoides. Phylogenetic relationship among all the plastotypes (plastid genotype) suggested the diphyletic origin of T. dicoccoides and T. araraticum. A plastotype of one A. speltoides accession was identical to the major type of T. araraticum (T. timopheevi inclusively). Three of the plastotypes found in the Sitopsis species are very similar, but not identical, to that of T. dicoccoides, T. durum and T. aestivum.

Chloroplasts↗
PubMed1992

Fine mapping of the McLeod locus (XK) to a 150-380-kb region in Xp21.

McLeod syndrome, characterized by acanthocytosis and the absence of a red-blood-cell Kell antigen (Kx), is a multisystem disorder involving a late-onset myopathy, splenomegaly, and neurological defects. The locus for this syndrome has been mapped, by deletion analysis, to a region between the loci for Duchenne muscular dystrophy (DMD) and chronic granulomatous disease (CGD). In this study, we describe a new marker, 3BH/R 0.3 (DXS 709), isolated by cloning the deletion breakpoint of a DMD patient. A long-range restriction map of Xp21, encompassing the gene loci for McLeod and CGD, was constructed, and multiple CpG islands were found clustered in a 700-kb region. Using the new marker, we have limited the McLeod syndrome critical region to 150-380-kb. Within this interval, two CpG-rich islands which may represent candidate sites for the McLeod gene were identified.

Blotting, Southern↗
PubMed1996

Spatial and temporal expression of 4f-rnp gene in Drosophila melanogaster.

We have recently discovered that a pre-messenger RNA (mRNA) encoded by the 4f-rnp gene in Drosophila melanogaster undergoes RNA editing by site-specific A-to-G conversions. In this report, we describe the spatial and temporal expression patterns of 4f-rnp mRNA transcripts during fly development. The 4f-rnp locus was mapped by polytene in situ hybridization experiments to the 4F1,2 bands at the distal tip of the X-chromosome, demonstrating that this gene is nuclear. We show that 4f-rnp is a single-copy gene by a combination of genomic Southern blot hybridization and restriction map analysis of clones isolated through chromosome walking techniques. Northern blot analysis indicates that two alternatively spliced messenger RNA transcripts, differing in length by 200-300 nucleotides, are synthesized in adult male and female flies, during embryogenesis and throughout the larval period. Using tissue in situ hybridization techniques that do not distinguish between the transcripts, we find that the distribution of 4f-rnp mRNAs is specific to germline tissue in the ovary, demonstrating that these transcripts are maternally produced. During embryogenesis, 4f-rnp transcripts are localized within cells of most tissues, but they have a very distinct localization pattern within the segmental ganglia of the central nervous system. We discuss the significance that RNA editing is expected to have for the predicted 4F-RNP protein products that may be expressed throughout fly development and in high abundance within the fly's central nervous system.

Alternative Splicing↗
PubMed1976

Physical map of Neurospora crassa mitochondrial DNA and its transcription unit for ribosomal RNA.

A circular denaturation and restriction map of mitochondrial DNA from Neurospora crassa is presented. The map shows the position of all twelve fragments produced by restriction endonuclease Eco R I and the position of the largest Hin III fragment along the previously established map of AT-rich sequences. The two wild type strains Em 5256 and 7A differ in the lengths of two Eco R I fragments. No difference was found between the mitochondrial mutant "poky" and its parent strain. The position of the DNA segment carrying the transcription unit for the two ribosomal RNA molecules has been determined by molecular hybridization.

Chromosome Mapping↗
PubMed1995

Loa loa: structural diversity of a 15-kDa repetitive antigen.

A ladder antigen of Loa loa was identified on Western blots of all life cycle stages probed with loaisis sera. The smallest subunit has a relative M(r) of about 15 kDa and larger subunits represent size increments of 15.0 kDa. An 1800-bp genomic clone encoding this antigen was characterized further by restriction mapping. Southern blot analysis, and nucleotide sequencing. The antigen is encoded by multiple copies of a gene, linked in tandem repeats of 396 bp, each of which encodes 132 amino acids. These repeats have diverged sufficiently to produce distinct restriction enzyme sites and Southern blot hybridization patterns. The 1764-bp insert contains no introns and encodes 588 amino acids, representing one incomplete and four complete repeats. At the 3' end of three repeats, there are consensus proteolytic cleavage sites; one repeat has no cleavage site. Two perfect repeats show a 93.9% amino acid identity with one another; the rest of the repeats, despite being adjacent to one another, show only 31-42% identical amino acids. Putative asparagine N-linked glycosylation sites are expressed by only two of the repeats. Despite this structural diversity, each L. loa repeat showed homology to Ascaris suum allergen and the homologue protein described in Dirofilaria immitis and Brugia pahangi.

Amino Acid Sequence↗
PubMed1991

Cloning and physical mapping of DNA sequences encompassing a region in N-myc amplicons of a human neuroblastoma cell line.

Cloning and physical mapping of DNA sequences encompassing N-myc amplicons of a human neuroblastoma cell line were done. A number of lambda phage clones within this region were isolated using the probes prepared by the phenol emulsion reassociation technique. Based on the restriction mapping, they were integrated into 8 contigs with sizes of 25-60 kb which, in total, encompassed a 330 kb region. Several amplicons, 100, 420, 480 and 520 kb in size as a Notl fragment, were identified using hexagonal field gel electrophoresis, and the contigs were assigned in these Notl fragments. The region encompassed by the contigs was equivalent to some 60-80% of the amplicons identified as a Notl fragment. In order to compare the amplified regions flanking the N-myc gene among the cell lines, the phage clones to cover the whole contigs were used for hybridization as a probe. The results showed that the portions of the whole contigs ranging 18-45% were also amplified in the cell lines examined. These results allowed us to identified the 'rearranged sites' which were rather evenly distributed, one at every 40 kb, through the contigs. These observations lead to the idea that an amplified DNA domain is constructed after the multiple rearrangements and then increases in number, finally resulting in the formation of subsets of amplicons with sequence homogeneity.

Cloning, Molecular↗
PubMed1990

A physical linkage group in human chromosome band 11q23 covering a region implicated in leukocyte neoplasia.

Six genes on human chromosome band 11q23 have been linked by pulse-field gel electrophoretic analysis with the order cen-CD3E-CD3D-CD3G-PBGD-CBL2-THY1-qter. The corresponding long-range restriction map covers 1.8 Mb, the telomeric half of which (PBGD-CBL2-THY1) is localized to subband 11q23.3. Four genes (CD3E, CD3D, CD3G, and PBGD) can be positioned precisely, and with known transcriptional orientation, with respect to rare-cutter restriction sites. The linkage group covers a region implicated in leukocyte malignancy: several nonrandom neoplasia-associated translocation chromosomes have breakpoints which separate the CD3 genes from PBGD, CBL2, and THY1. Thus, we are able to localize such breakpoints, and consequently any affected candidate genes, to the 750 kb between CD3G and PBGD.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Rearrangement of VHa1-encoding Ig gene segment to the a2 chromosome in an a1/a2 heterozygous rabbit. Evidence for trans recombination.

VDJ genes were cloned from leukemic B cells of an a1/a2 heterozygous Emu-cmyc transgenic rabbit. Restriction mapping and nucleotide sequence analysis indicated that one clone, 5C3, had a VHa1-encoding gene segment functionally rearranged to a JH gene segment from the a2 chromosome. This VDJ gene may be the result of a trans recombination between a VH gene on the a1 chromosome and a JH gene segment on the a2 chromosome or, it may be the result of a cis recombination if the a2 chromosome contains VHa1-encoding gene segments.

Amino Acid Sequence↗
PubMed1994

Evaluation of the gene encoding the gamma subunit of rod phosphodiesterase in retinitis pigmentosa.

PURPOSE: To determine whether defects in the gene encoding the gamma subunit of rod cyclic guanosine monophosphate-phosphodiesterase (PDE-g) cause some form of hereditary retinal degeneration or dysfunction. METHODS: A restriction map, an intron/exon map, and a partial sequence of the human genomic locus corresponding to this gene were ascertained. Based on this information, the single-strand conformation polymorphism technique (SSCP) was used to screen the coding region as well as most splice donor and acceptor sites for mutations in a total of 704 unrelated patients with retinitis pigmentosa, Usher's syndrome type I or type II, Leber's congenital amaurosis, the Laurence-Moon-Bardet-Biedl syndrome, or other hereditary retinal disease. RESULTS: Two frequent polymorphisms were found, as well as three rare sequence variations, none of which correlated with any phenotype examined. CONCLUSIONS: In view of these negative results and those of a previously published negative Southern blot analysis of an overlapping set of patients, it is unlikely that mutations in the PDE-g gene are a common cause of any of the forms of retinal degeneration or dysfunction so far examined.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1996

Characterization of Vibrio cholerae EIT or typing phage D10.

The Vibrio cholerae EITor typing phage D10 was characterized. The adsorption kinetics of the phage on V. cholerae MAK757 strain were biphasic in nature. Intracellular growth was characterized by an eclipse period, latent period and burst size which were 20 min, 25 min and 80 particles per cell respectively. The phage yield was dependent on the concentration and time of addition of DNA synthesis inhibitors such as nalidixic acid and novobiocin, and RNA synthesis inhibitors such as rifampicin. The 32+/-0.2 kb linear double-stranded DNA molecule has unique termini. A restriction map of the phage DNA was constructed with the enzymes BamHI, HindIII and PstI.

Adsorption↗
PubMed1997

Physical mapping of the murine casein locus reveals the gene order as alpha-beta-gamma-epsilon-kappa.

The murine casein locus has been characterized by long-range restriction mapping and the analysis of large fragment genomic clones. Cloned sequences from five mouse casein genes (alpha, beta, gamma, epsilon, kappa) were used to screen a murine (strain 129) genomic library in a bacterial artificial chromosome vector (BAC). Of the nine clones isolated, two contained three casein genes alpha, beta, gamma and gamma, epsilon, kappa, respectively. The following combinations were found in other clones: alpha + beta, beta + gamma; and gamma + epsilon. Thus, the gene order in the locus can be deduced to be alpha-beta-gamma-epsilon-kappa. This order was confirmed by restriction analysis of the clones. A contig map of the clones and flanking sequences has been established by characterizing seven BAC clones, which together span approximately 470 kb. Long-range restriction analysis of genomic DNA indicates that the murine casein locus is confined to a 250-kb partial Xho I fragment. The alpha and beta casein genes were shown to be arranged in a tail-to-tail orientation.

Animals↗
PubMed1989

Altered restriction pattern of the putative DNA binding domain of estrogen receptor or related genes in primary human meningiomas.

We analyzed by restriction mapping the genomic organization of the estrogen receptor gene in several primary human brain tumors in order to investigate the possible relationships between the development of these tumors and gonadal steroid hormones. In 5 out of 23 meningiomas the Eco RI restriction of genomic DNAs revealed 6 invariant normal fragments of 6.5, 4.8, 3.8, 3.1, 2.7 and 1.7 kb, plus 2 additional variant fragments of either 8.6 or 2.5 kb. More detailed analysis showed that these variant bands hybridized with a probe specific for the middle region of the estrogen receptor cDNA, coding for the DNA-binding domain of the receptor. This abnormal restriction pattern was found only in these meningiomas and not in other brain tumors or DNAs obtained from peripheral blood lymphocytes.

Blotting, Southern↗
PubMed1990

Physical mapping and molecular cloning of mung bean yellow mosaic virus DNA.

Viral single-stranded DNA of mung bean yellow mosaic virus (MYMV) was converted to the double-stranded state in vitro, and physical mapping was carried out. The genome of MYMV was found to consist of two major components (designated as DNA 1 and DNA 2). In addition, some minor components were detected. Molecular cloning of the major components was carried out, using in vitro double-stranded DNA and replicative intermediate DNAs. DNA 1 is about 2.72 and DNA 2 about 2.67 kilobase pairs. No similarities were observed when the two restriction maps of DNA 1 and 2 were compared.

Cloning, Molecular↗
PubMed1998

Precise mapping and characterization of the RNA primers of DNA replication for a yeast hypersuppressive petite by in vitro capping with guanylyltransferase.

The active origins of DNA replication for yeast (Saccharomyces cerevisiae) mitochondrial DNA share 280 conserved base pairs and have a promoter. Since intact replication intermediates retain their initiating ribonucleotide triphosphate, we used guanylyltransferase to in vitro cap the replication intermediates present in restriction enzyme-cut DNA from an ori-5 hypersuppressive petite. Restriction mapping and RNA sequencing of these labeled intermediates showed that each DNA strand is primed at a single discrete nucleotide, that one primer starts at the promoter and that the other primer starts 34 nt away, outside the conserved region. Deoxyribonuclease digestion of the capped fragments left resistant RNA primers, which enabled identification of zones of transition from RNA to DNA synthesis. Some of the results contradict the prevailing model for priming at the yeast mitochondrial origins.

Base Sequence↗
PubMed1996

Characterization and comparison of the human and mouse Dist1/alpha-globin complex reveals a tightly packed multiple gene cluster containing differentially expressed transcription units.

In this paper, we describe the detailed analysis of about 75 kb of genomic DNA flanking the 5' end of the mouse alpha-globin region and complete the transcription map of the human region. Previously, we established the homology of the human and mouse alpha-globin upstream flanking regions (alpha UFR) and characterized in detail the mouse alpha-globin major regulatory element (alpha MRE) and the mMPG DNA repair gene. Here, we extend our analysis with the construction of a detailed restriction map, the mapping and isolation of two nonglobin genes, named mDist1 and mProx1, the distribution of 18 DNase hypersensitive sites (HSSs) in erythroid and fibroblast cells, and the analysis of the mDist1, mMPG, and mProx1 expression levels in several adult tissues and during fetal development. In addition, the hDist1 gene is exactly localized 1.9 kb from the hMPG gene. The mapping results show that the Dist1, MPG, and Prox1 genes, together with the alpha-globin genes and the alpha MRE, form a tightly packed multiple gene cluster that is 50% more compact in mouse than in human. The expression results show that each of the genes present in this locus displays a characteristic expression pattern in adult tissues and during fetal development. The 18 DNase HSSs observed were scattered over this region. Interestingly, all the erythroid-sensitive HSSs were associated with the Prox1 transcription unit, whereas the only two pairs of fibroblast-sensitive HSSs present in this locus were located in the promoter regions of the mProx1 and mDist1/mMPG genes. The possible role of the erythroid- and fibroblast-sensitive sites in the regulation of the mouse alpha-globin and nonglobin gene expression is discussed. The characterization of the mouse alpha UFR identifies most, if not all, of the structural elements possibly involved in the regulation of m alpha-globin gene expression and sheds light on the organization and evolution of the telomere-associated, GC-rich isochore family H3.

Animals↗
PubMed1994

[Mapping of full-length murine renin-2 gene].

A 24kb fragment containing full-length renin-2 gene and its flanking sequence was obtained by digesting the pKG-R2D with XhoI. This fragment was cleaved with single enzyme (EcoRI, KpnI and BamHI), and combined enzymes (EcoRI/KpnI, KpnI/BamHI and BamHI/EcoRI), respectively. The digests were electrophoresed on 0.8% agarose plates and transferred onto NC membranes. Radioactive probes of 735bp and 1400bp, templates of which were from half and full-length of renin-1 cDNA, respectively were used for Southern hybridization. On the basis of electrophoresis and hybridization patterns, a restriction map was constructed in this paper.

Animals↗
PubMed1996

A 1.6-Mb P1-based physical map of the Down syndrome region on chromosome 21.

The Down syndrome (DS) region on chromosome 21, which is responsible for the main features of DS such as characteristic facial features, a congenital heart defect, and mental retardation, has been defined by molecular analysis of DS patients with partial trisomy 21. The 2. 5-Mb region around the marker D21S55 between D21S17 and ERG in 21q22 is thought to be important, although contributions of other regions cannot be excluded. In this region, we focused on a 1.6-Mb region between a NotI site, LA68 (D21S396, which is mapped distal to D21S17) and ERG, because analysis of a Japanese DS family with partial trisomy 21 revealed that the proximal border of its triplicated region was distal to LA68. We constructed P1 contigs with 46 P1 clones covering more than 95% of the 1.6-Mb region. A high-resolution restriction map using BamHI was also constructed for more detailed analysis. Our P1 contig map supplements other physical maps previously reported and provides useful materials for further analysis including gene isolation and sequencing of the DS region.

Base Sequence↗
PubMed1991

Large inversion in Escherichia coli K-12 1485IN between inversely oriented IS3 elements near lac and cdd.

A companion study has shown that the inversion carried by strain 1485IN has one terminus between lac and proC and the other between his and cdd of the normal strain. Starting with this mapping data, we have done molecular work demonstrating that the inversion occurred by recombination between inversely oriented two IS3 elements, one present near lac and the other near the cdd locus; i.e., the inversion is IN(is3B-is3E). Evidence supporting this conclusion includes: (i) Normal and inversion strains share two short regions with identical restriction maps. One of these regions is near lac and the other near cdd. (ii) IS3 homology was detected in each of the terminus regions of both the normal and inversion strains. (iii) The sequence on one side of the original IS3 element near lac has been exchanged with the sequence on one side of the IS3 near cdd. Whether the inversion has occurred by one event of homologous recombination between the two IS3 elements or has been caused by involvement of IS3 elements on an F factor is discussed. Another rearrangement, probably related to inversion and deletion, was detected between the IS3 and cdd of the inversion strain.

Base Sequence↗