Polyoma virus defective DNAs. II. Physical map of a molecule with rearranged and reiterated sequences (D74).

The structure of the polyoma virus defective species D74 (74% the size of full-length polyoma virus DNA) has been determined and compared with that of polyoma virus A2 DNA. D74 appears to be composed entirely of viral DNA sequences. (No host DNA sequences have been detected.) It is made up of three DNA segments, each about 24, 24 and 27% in size. The two 24% segments appear to be identical and the 27% segment contains one copy of all the sequences found in the 24% fragments as well as a duplication of some of the sequences. When related to the physical map of A2 DNA, each segment is found to be composed of viral sequences from 1 to about 19 map units, 67 to 69 map units and 70 to 72 map units.Three features found in other polyoma virus defective species (Lund et al., 1977) are also present in D74. (1) Sequences from the region around 67 map units are linked to other (non-contiguous) viral sequences. (2) Sequences at about 72 map units are linked to sequences at 1 map unit. (3) Multiple copies of sequences from around the origin of viral DNA replication are present. From studies on other polyoma defective molecules (Griffin &; Fried, 1975; Lund et al., 1977), the origin of DNA replication for polyoma virus has been defined to lie within the sequences from 67 to 72 map units. Since D74 replicates efficiently but lacks the sequences between 69 to 70 map units, the origin of DNA replication appears to be further defined as lying within 67 and 69 map units and/or 70 to 72 map units.     

Polyoma virus defective DNAs. I. Physical maps of a related set of defective molecules (D76, D91, D92).

Three related polyoma virus species, designated D92 (92% the size of full-length polyoma virus DNA), D91 (91%) and D76 (76%) have been analysed and their structures compared with that of polyoma virus A2 DNA. Three independent methods (restriction endonuclease cleavage, depurination fingerprinting and DNA-DNA hybridization) were used in the analysis.The defective DNAs appear to be: (1) entirely composed of viral sequences (no host DNA sequences were detected): (2) made up in part of long continuous sequences of DNA which appear identical to sequences of A2 DNA (D92 contains continuous sequences from 1 to 72 map units on the physical map of A2 DNA; that is, it contains the entire late region and part of the early region of the viral DNA. D91 and D76 contain those same sequences except for a 1% deletion around 18 map units): (3) made up in part of rearranged viral sequences.Several interesting features were noted about the rearranged sequences present in the defective DNAs. Sequences from the region around 67 map units were found linked to other (non-contiguous) regions of the DNA. Sequences from about 72 map units were linked to sequences from about 1 map unit. Multiple copies of sequences from 67 to 72 map units (from around the origin of DNA replication) were found (4 copies in D91 and D92, and 2 copies in D76).     

Differentiation between wheat chromosomes 4B and 4D.

A linkage map based on homoeologous recombination, induced by the absence of the Ph1 locus, between chromosome 4D of Triticum aestivum L. (genomes AABBDD) and chromosome 4B of T. turgidum L. (genomes AABB) was compared with a linkage map of chromosome 4Am of T. monococcum L. and a consensus map of chromosomes 4B and 4D of T. aestivum based on homologous recombination. The 4D/4B homoeologous map was only one-third the length of the homologous maps and all intervals were reduced relative to the 4B-4D consensus map. After the homoeologous map was corrected for this overall reduction in recombination, the distribution of recombination in the short arm was similar in both types of maps. In the long arm, homoeologous recombination declined disproportionally in the distal to proximal direction. This gradient was shown to be largely caused by severe segregation distortion reflecting selection against 4D genetic material. The segregation distortion had a maximum that coincided with the centromere and likely had a polygenic cause. Chromosomes 4D and 4B were colinear and recombination between them occurred in almost all intervals where homologous recombination occurred. These findings suggest that these chromosomes are not differentiated structurally and that the differentiation is not segmental. In the presence of Ph1, metaphase I chromosome pairing between chromosomes composed of homologous and differentiated regions correlated with the lengths of the homologous regions. No compensatory allocation of crossovers into the homologous regions was detected. In this respect, the present results are in dramatic contrast with the crossover allocation into the pseudoautosomal region in the mammalian male meiosis.     

A cytogenetically based physical map of chromosome 1B in common wheat.

We have constructed a cytogenetically based physical map of chromosome 1B in common wheat by utilizing a total of 18 homozygous deletion stocks. It was possible to divide chromosome 1B into 17 subregions. Nineteen genetic markers are physically mapped to nine subregions of chromosome 1B. Comparison of the cytological map of chromosome 1B with an RFLP-based genetic linkage map of Triticum tauschii revealed that the linear order of the genetic markers was maintained between chromosome 1B of hexaploid wheat and 1D of T. tauschii. Striking differences were observed between the physical and genetic maps in relation to the relative distances between the genetic markers. The genetic markers clustered in the middle of the genetic map were physically located in the distal regions of both arms of chromosome 1B. It is unclear whether the increased recombination in the distal regions of chromosome 1B is due to specific regions of increased recombination or a more broadly distributed increase in recombination in the distal regions of Triticeae chromosomes.     

Addition of MT, D16S10, D16S4, and D16S91 to the linkage map within 16q12.1-q22.1.

A 10-point genetic linkage map of the region 16q12.1 to 16q22.1 has been constructed using the CEPH reference families. Four loci, MT, D16S10, D16S91, and D16S4, not previously localized on a multipoint linkage map, were incorporated on the map presented here. The order of loci was cen-D16S39-MT, D16S65-D16S10-FRA16B-D16S38, D16S4, D16S91, D16S46-D16S47-HP-qter. The interval between D16S10 and 4D16S38 is 3.1 cM in males and 2.3 cM in females, and contains FRA16B. The cloning strategy for FRA16B will now be based on YAC walking from D16S10 and D16S38. The location of FRA16B between D16S10 and D16S38 provides a physical reference point for the multipoint linkage map on the short arm of chromosome 16.     

Mapping of multiple B cell epitopes on the 70-kilodalton autoantigen of the U1 ribonucleoprotein complex.

High titer IgG autoantibodies to the 70-kDa polypeptide component (p70) of the U1 ribonucleoprotein (RNP) complex occur in the sera of patients with mixed connective tissue disease, SLE, and related rheumatic diseases. To gain insight into the pathogenesis and diversity of this antibody response we have used recombinant DNA technology to map the linear B cell epitopes on p70. A full length 1.7-kb cDNA clone encoding p70 was isolated from a human placental library and restriction fragments or polymerase chain reaction-generated fragments of the gene subcloned into the bacterial expression vector pGEX. Purified fusion proteins representing specific regions of p70 were immunoblotted with a panel of 70 anti-(U1)RNP+ sera containing anti-p70 antibodies. Six epitopes, four major (A, B, C, and F) and two minor (D and E) were mapped and were located throughout the molecule. The anti-(U1)RNP sera displayed heterogeneity in their pattern of reactivity to the six epitopes although reactivity to epitope C was more frequently associated with SLE rather than mixed connective tissue disease. The identification of multiple B cell epitopes on p70 is consistent with the concept that this self Ag drives the autoantibody response.     

Genome assembly and annotation of a Drosophila simulans strain from Madagascar

Drosophila simulans is a close relative of the genetic model D. melanogaster. Its worldwide distribution in combination with the absence of segregating chromosomal inversions makes this species an increasingly attractive model to study the molecular signatures of adaptation in natural and experimental populations. In an effort to improve the genomic resources for D. simulans, we assembled and annotated the genome of a strain originating from Madagascar (M252), the ancestral range of D. simulans. The comparison of the M252 genome to other available D. simulans assemblies confirmed its high quality, but also highlighted genomic regions that are difficult to assemble with NGS data. The annotation of M252 provides a clear improvement with alternative splicing for 52% of the multiple‐exon genes, UTRs for 70% of the genes, 225 novel genes and 781 pseudogenes being reported. We anticipate that the M252 genome will be a valuable resource for many research questions.     

Genetic mapping of 14 short tandem repeat polymorphisms on human chromosome 22

We have constructed a linkage map of 14 short tandem repeat polymorphisms (11 with heterozygosity > 70%) on the long arm of human chromosome 22 using 23 non-CEPH pedigrees. Twelve of the markers could be positioned uniquely with a likelihood of at least 1,000:1, and distributed at an average distance of 6.62 cM (range 1.5–16.1 cM). The sex-combined map covers a total of 79.6 cM, the female map 93.2 cM and the male map 64.6 cM. Based on comparisons between physical maps and other genetic maps, we estimate that our map covers 70%–80% of the chromosome. The map integrates markers from previous genetic maps and uniquely positions one marker (D22S307). Data from physical mapping on the location of four genetic markers correlates well with our linkage map, and provides information on an additional marker (D22S315). This map will facilitate high resolution mapping of additional polymorphic loci and disease genes on chromosome 22, and act as a reference for building and verifying physical maps.     

QTL mapping of 1000-kernel weight, kernel length, and kernel width in bread wheat (Triticum aestivum L.)

Kernel size and morphology influence the market value and milling yield of bread wheat (Triticum aestivum L.). The objective of this study was to identify quantitative trait loci (QTLs) controlling kernel traits in hexaploid wheat. We recorded 1000-kernel weight, kernel length, and kernel width for 185 recombinant inbred lines from the cross Rye Selection 111 × Chinese Spring grown in 2 agro-climatic regions in India for many years. Composite interval mapping (CIM) was employed for QTL detection using a linkage map with 169 simple sequence repeat (SSR) markers. For 1000-kernel weight, 10 QTLs were identified on wheat chromosomes 1A, 1D, 2B, 2D, 4B, 5B, and 6B, whereas 6 QTLs for kernel length were detected on 1A, 2B, 2D, 5A, 5B and 5D. Chromosomes 1D, 2B, 2D, 4B, 5B and 5D had 9 QTLs for kernel width. Chromosomal regions with QTLs detected consistently for multiple year-location combinations were identified for each trait. Pleiotropic QTLs were found on chromosomes 2B, 2D, 4B, and 5B. The identified genomic regions controlling wheat kernel size and shape can be targeted during further studies for their genetic dissection.     

Electron microscopic analysis of the E polytene chromosome of Drosophila subobscura: divisions 57, 58 and 59

The banding pattern of the divisions 57, 58 and 59 of the E polytene chromosome of Drosophila subobscura was analyzed by electron microscopy. Using squashed and thin-sectioned polytene chromosomes, our electron microscopic results have been compared with the reference map of Kunze-Müller (Chromosoma 9, 559-570 (1958]. These divisions are rich in heavy bands, and their number and location coincide with those of the reference map. The major differences observed between our electron micrographs and the reference map have been at the level of faint bands.     

Mouse X chromosome

Overall, the probe map fromDXWas70 toAmg encompasses 72 cM and includes 103 loci. Eight of these have been designated reference loci (see Table 2 and previous section) on account of their wide usage that would enable the cross reference of independent maps created in different laboratories. Reference loci are to be readily available and easily used probes for Southern hybridization. By and large, they will be STSs, regeneratable by PCR, and correspond to a known gene. In addition, on the mouse X Chr, there is a reference locus from each of the known conserved linkage groups found between the mouse and human X Chrs. All the loci, barDXWas31, represent conserved sequences. Committee Members: D. Adler, P.J. Barnard, Y. Boyd, N. Brockdorff, J. Derry, C. Disteche, C. Faust, M.F. Lyon, S. Rastan, M. Seldin and L. Siracusa.     

An update of the Courtot × Chinese Spring intervarietal molecular marker linkage map for the QTL detection of agronomic traits in wheat

We made an update of the intervarietal molecular marker linkage map of the wheat genome developed using a doubled-haploid (DH) population derived from the cross between the cultivars "Courtot" and "Chinese Spring". This map was constructed using 187 DH lines and 659 markers. The genome was well covered (more than 95%) except for chromosomes from homoeologous group 4 and chromosomes 5D and 7D, which had gaps slightly larger than 50 cM. A core-map based on a set of 200 anchor loci (one marker each 18.4 cM) was developed. The total length of this map was 3,685 cM which is similar to the size of the international reference map of the ITMI population (3,551 cM). Map coverage was identical for the three genomes (A, B and D) and for the number of anchor loci, as well as for the size of the map. Using this map, QTLs for several agronomic traits were detected on phenotypic data from the population grown in Clermont-Ferrand (France) under natural field conditions over 6 years, and in Norwich (UK) in controlled conditions and under natural field conditions in 1 year. Almost all of the 21 chromosomes were involved in at least one trait. However, several regions seemed to contain gene clusters either for grain traits (and thus bread-making quality) or plant development traits.     

Comparison of genetic and physical maps of group 7 chromosomes from Triticum aestivum L.

We present a high density physical map of homoeologous group 7 chromosomes from Triticum aestivum L. using a series of 54 deletion lines, 6 random amplified polymorphic DNA (RAPD) markers and 91 cDNA or genomic DNA clones from wheat, barley and oat. So far, 51 chromosome segments have been distinguished by molecular markers, and 54 homoeoloci have been allocated among chromosomes 7A, 7B and 7D. The linear order of molecular markers along the chromosomes is almost identical in the A- B- and D-genome of wheat. In addition, there is colinearity between the physical and genetic maps of chromosomes 7A, 7B and 7D from T. aestivum, indicating gene synteny among the Triticeae. However, comparison of the physical map of chromosome 7D from T. aestivum with the genetic map from Triticum tauschii some markers have been shown to be physically allocated with distortion in more distal chromosome regions. The integration of genetic and physical maps could assist in estimating the frequency and distribution of recombination in defined regions along the chromosome. Physical distance did not correlate with genetic distance. A dense map facilitates the detection of multiple rearrangements. We present the first evidence for an interstitial inversion either on chromosome arm 7AS or 7DS of Chinese Spring. Molecularly tagged chromosome regions (MTCRs) provide landmarks for long-range mapping of DNA fragments.     

Thermosensitive Bacillus subtilis mutants which lyse at the non-permissive temperature

A collection of 655 thermosensitive mutants of Bacillus subtilis 168, obtained by indirect selection, was screened for those lysing at the non-permissive temperature. Thirty-three mutations thus identified were distributed by transformation into eight linkage groups designated lssA to lssH. The distribution was non-random. With the exception of group A, all groups were small, suggesting that mutations identified in each of them may map in one gene only. Linkage groups identified here were mapped in four different regions of the B. subtilis chromosome and their positions relative to reference markers were the following: (i) aroI-lssA-dal-purB; (ii) metC-lssB-lssC-furA-pyrE-cysC-lssD; (iii) lssF-gtaA-lssG-hisA-lssH-cysB; and (iv) cysA-lssE-dnaC-purA. Kinetics of N-acetyl-D-[1-14C]glucosamine incorporation revealed that groups A, B, C, D and F are deficient in peptidoglycan synthesis at the restrictive temperature. In group G, anomalies at the cell wall level were suggested by incorporation and growth curves. It appears that in almost all known cases, thermosensitive lysis mutations in B. subtilis either affect genes involved in peptidoglycan synthesis or lead, more or less directly, to induction of prophages.     

Mapping protein-protein interactions with a library of tethered cutting reagents: the binding site of sigma 70 on Escherichia coli RNA polymerase

Surface-exposed lysine amino groups and other reactive nucleophiles of the sigma 70 protein were conjugated with the cutting reagent iron (S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (FeBABE) via 2-iminothiolane (2IT) with low efficiency. The result is a library of sigma 70 conjugates, with an average of 1-2 cutting reagents tethered to any of a variety of sites (lysine, cysteine, etc.) on the surface of the protein. Model calculations indicate that the conjugates in this library should be capable of cutting nearby sites on the backbone of almost any protein or nucleic acid to which sigma 70 binds. Since cutting occurs only when the protein is bound, the cleaved sites indicate proximity; since only proximal sites are cleaved, interpretation of the results is straightforward. We used this library to map the periphery of the binding site on the core enzyme (alpha 2 beta beta') of Escherichia coli RNA polymerase. The beta subunit was cut primarily within its conserved regions C, D, Rif I, and G; additional sites were also cut between A and B and near conserved regions E and H. The cut sites within the beta' subunit were intensely clustered between residues 250-450, which include its conserved regions C and D, along with two additional cut sites in conserved regions A and G. No cut sites on the alpha subunit were observed. These results recapitulate and extend those obtained using FeBABE conjugates of seven strategically placed single-Cys sigma 70 mutants [Owens, J. T., Miyake, R., Murakami, K., Chmura, A. J., Fujita, N., Ishihama, A., and Meares, C. F. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6021-6026]. This technique provides a straightforward, general approach to mapping protein interactions without mutagenesis.     

1A and 3D gene sequences of coxsackievirus B3 strain CC: variation and phylogenetic analysis.

Coxsackievirus B3 (CVB3) was thought to be the most common causative agent of life-threatening viral myocarditis. Coxsackievirus B3 strain CC (CVB3-CC) was isolated in China; however, no sequence data are available. The 1A and 3D regions of CVB3-CC were sequenced and phylogenetic analysis was done with reference to ten other CVB3 strains and all 36 prototype strains of human enterovirus B (HEV-B). Sequence analysis showed that the 1A gene region of CVB3-CC consisted of 207 nucleotides, encoding 69 amino acids; and the 3D gene region was comprised of 1386 nucleotides, encoding 462 amino acids. Variation analysis showed that the 3D gene of CVB3 strain CC varied the least among the two regions. Phylogenetic tree analysis of the 1A and 3D regions indicated that CVB3-CC clustered together with CVB3 Nancy strain suggesting that there may be a close evolutionary relationship between the two strains. Incongruity was observed between the non-structural protein gene and the structural protein gene trees, according to the topological structure, indicating that recombination was occurred among these strains.     

Surface expression of Hsp70B’ in response to proteasome inhibition in human colon cells

Hsp70B’ was expressed on the surface of HT-29 and CRL-1809 but not SW-480 human colon cell lines in response to proteasome inhibition as detected using flow cytometry. Surface expression was not detected under non-stress conditions nor was heat shock an inducer of surface expression in the three cell lines tested. Phylogenetic analysis indicated that the Hsp70B’ protein sequence was most closely related to another major inducible human Hsp70, Hsp72. Hsp70B’ appeared to be recently diverged, as homologs for Hsp70B’ have not been found in rodents. Hsp72 and Hsp70B’ shared 100% amino acid sequence identity in their predicted peptide-binding regions suggesting that they bind the same peptide substrates, perhaps in extracellular antigen presentation. Amino acid sequence differences were concentrated in the lid regions and the C-terminal domains raising the possibility that Hsp72 and Hsp70B’ bind different co-chaperones or cell surface receptors.     

A chromosome bin map of 16,000 expressed sequence tag loci and distribution of genes among the three genomes of polyploid wheat

Because of the huge size of the common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) genome of 17,300 Mb, sequencing and mapping of the expressed portion is a logical first step for gene discovery. Here we report mapping of 7104 expressed sequence tag (EST) unigenes by Southern hybridization into a chromosome bin map using a set of wheat aneuploids and deletion stocks. Each EST detected a mean of 4.8 restriction fragments and 2.8 loci. More loci were mapped in the B genome (5774) than in the A (5173) or D (5146) genomes. The EST density was significantly higher for the D genome than for the A or B. In general, EST density increased relative to the physical distance from the centromere. The majority of EST-dense regions are in the distal parts of chromosomes. Most of the agronomically important genes are located in EST-dense regions. The chromosome bin map of ESTs is a unique resource for SNP analysis, comparative mapping, structural and functional analysis, and polyploid evolution, as well as providing a framework for constructing a sequence-ready, BAC-contig map of the wheat genome.     

Homology of polytene elements between Drosophila and Zaprionus determined by in situ hybridization in Zaprionus indianus

The drosophilid Zaprionus indianus due to its economical importance as an insect pest in Brazil deserves more investigation into its genetics. Its mitotic karyotype and a line-drawing map of its polytene chromosomes are already available. This paper presents a photomap of Z. indianus polytene chromosomes, which was used as the reference map for identification of sections marked by in situ hybridization with gene probes. Hybridization signals for Hsp70 and Hsr-omega were detected, respectively, in sections 34B and 32C of chromosome V of Z. indianus, which indicates its homology to the chromosomal arm 3R of Drosophila melanogaster and, therefore, to Muller's element E. The main signal for Hsp83 gene probe hybridization was in section 17C of Z. indianus chromosome III, suggesting its homology to arm 3L of D. melanogaster and to element D of Muller. The Ubi probe hybridized in sections 10C of chromosome II and 17A of chromosome III. Probably the 17A is the polyubiquitin locus, with homology to arm 3L of D. melanogaster and to the mullerian D element, as suggested also by Hsp83 gene location. The Br-C gene was mapped in section 1D, near the tip of the X chromosome, indicating its homology to the X chromosome of D. melanogaster and to mullerian element A. The Dpp gene probe hybridized mainly in the section 32A of chromosome V and, at lower frequencies to other sections, although no signal was observed as expected in the correspondent mullerian B element. This result led to the suggestion of a rearrangement including the Dpp locus in Z. indianus, the secondary signals possibly pointing to related genes of the TGF-beta family. In conclusion, the results indicate that chromosomes X, III, V of Z. indianus are respectively correspondents to elements A, D, and E of Muller. At least chromosome V of Z. indianus seems to share synteny with the 3R arm of D. melanogaster, as indicated by the relative positions of Hsp70 and Hsr-omega, although the Dpp gene indicates a disruption of synteny in its distal region.     

Mapping Bombina mitochondrial genomes: the conundrum of Carpathian Bombina variegata (Anura: Discoglossidae)

Complete mitochondrial genomes (mtDNA) of five individuals representing two haplotypes of Bombina bombina and three of Bombina variegata were compared using restriction site maps. Phylogenetic analyses reveal three ancient mitochondrial lineages: (1) two very similar haplotypes A and B of B. bombina ; (2) almost identical haplotypes D and E of B. variegata ; and (3) haplotype C of B. variegata . Haplotype C is as different from haplotypes D/E as from A/B. These data are strikingly discordant with relationships based on morphology and allozymes. Haplotypes C and D/E represent a pre-Pleistocene mitochondrial divergence within B. variegata , nearly coincident with speciation between B. variegata and B. bombina . Geographical partitioning of the two divergent B. variegata mitochondrial lineages indicates repeated localization of the lineages in separate glacial refugia during the Pleistocene. That nuclear genes do not show a similar divergence, but rather indicate relatively free genetic exchange between populations with divergent mtDNAs, suggests that males dispersed much more widely than females during expansions from glacial refugia. Comparison of Bombina mtDNA maps with a restriction site map of Xenopus laevis mtDNA revealed 16 homologous sites; 12 of these may be nearly invariant across primitive anuran mtDNAs. Two distinct regions of heteroplasmy, representing two regions with variable numbers of sequence repeats [length variable (LV) regions], were characterized. Comparison with the Xenopus map places LV1, present in all five haplotypes, near the 5'-end of the control region, and LV2 present only in B. variegata , near the 3'-end. Although phylogenetic analyses did not group the two major B. variegata lineages together, presence of LV2 in both lineages supports placement of both within B. variegata .