Map Of DNA Research Articles

Human chromosomal bands: nested structure, high-definition map and molecular basis

In this paper, we report investigations on the nested structure, the high-definition mapping, and the molecular basis of the classical Giemsa and Reverse bands in human chromosomes. We found the rules according to which the approximately 3,200 isochores of the human genome are assembled in high (850-band) resolution bands, and the latter in low (400-band) resolution bands, so forming the nested mosaic structure of chromosomes. Moreover, we identified the borders of both sets of chromosomal bands at the DNA sequence level on the basis of our recent map of isochores, which represent the highest-resolution, ultimate bands. Indeed, beyond the 100-kb resolution of the isochore map, the guanine and cytosine (GC) profile of DNA becomes turbulent owing to the contribution of specific sequences such as exons, introns, interspersed repeats, CpG islands, etc. The isochore-based level of definition (100 kb) of chromosomal bands is much higher than the cytogenetic definition level (2-3 Mb). The major conclusions of this work concern the high degree of order found in the structure of chromosomal bands, their mapping at a high definition, and the solution of the long-standing problem of the molecular basis of chromosomal bands, as these could be defined on the basis of compositional DNA properties alone.

Restriction mapping of Cucurbitae pepo L. chloroplast DNA Pstl 9.8 kb fragment and detection of ORF 2280 transcripts

Using tomato heterologous probe, the ORF 2280 was located on the PstI 9.8 kb fragment in the inverted repeat A (IRA) and on the PstI 12.1 kb fragment in the inverted repeat B (IRB) on the physical map of squash chloroplast DNA. A fine map of the PstI 9.8 kb fragment was developed using SalI, BamHI and EcoRI restriction enzymes.Two transcripts of 1.4and 2.6 kbs of ORF 2280 of squash plastid DNA were detected in fruits but not in the leaves of Early Prolific cultivar using a heterologous PstI 1.2 kb tomato plastid DNA probe internal to ORF 2280.In Bicolor gourd fruits, 1.4 and 2.6 kb transcripts of ORF 2280 weredetected both in the fruits and in leaves. Key Words: Cucurbitae pepo, restriction mapping, PstI 9.8 kb fragment, ORF 2280 transcripts. African Journal of Biotechnology Vol.4(1) 2005: 1-8

Pronounced chromosomal instability and multiple gene amplifications characterize ulcerative colitis–associated colorectal carcinomas

Patients with ulcerative colitis have a significantly increased lifetime risk for the development of colorectal carcinomas. While genetic and genomic changes during carcinogenesis have been thoroughly studied in sporadic colorectal cancers, less is known about ulcerative colitis–associated colorectal carcinomas. The aim of this study was to extend the identification of specific genomic imbalances to ulcerative colitis–associated colorectal carcinomas and to establish a comprehensive map of DNA gains and losses by investigating 23 tumor specimens from 23 patients. The molecular cytogenetic characterization was performed using comparative genomic hybridization; immunohistochemistry was used to measure proliferative activity and laminin-5 expression as a marker for invasiveness. The results indicate that these tumors are invariably aneuploid, with a high proliferative activity and increased invasive potential. The average number of copy alterations correlates with increased cyclin A levels ( P = 0.044), which is an independent predictor of risk of carcinoma development in ulcerative colitis. Despite severe genetic instability, the general pattern of specific chromosomal aberrations that defines sporadic colorectal carcinomas is maintained in ulcerative colitis–associated malignancies. High-level copy number increases (amplifications) are dispersed throughout the genome. Strikingly, these amplifications are much more frequent than in sporadic carcinomas and map to chromosomal regions that have not been described before.

The mitochondrial genome of Debaryomyces (Schwanniomyces) occidentalis encodes subunits of NADH dehydrogenase complex I

nad genes encoding subunits of the NADH dehydrogenase complex 1 have been revealed in the yeast Debaryomyces (Schwanniomyces) occidentalis. nad1, nad3, nad5, nad6 and most large mitochondrial genes have been located on a circular 41-kb map of mitochondrial DNA from this petite negative species. The genes nad1– nad6 are co-transcribed and the transcription is not inhibited by glucose. Sequences of nad6 and 5′ -nad1 compared to homologs in other yeasts indicate better amino acids conservation for nad1 product than for nad6. A cytochrome b deficient mutant dependent on alternative oxidase and functional complex 1 for growth on respirable substrates also exhibits co-transcription of nad1– nad6.

Jumping mode scanning force microscopy: a suitable technique for imaging DNA in liquids

In this work, we introduce jumping mode (JM) scanning force microscopy (SFM) as a suitable technique for imaging soft samples in liquid environment like DNA adsorbed on mica. JM reveals as a non-intrusive technique where shear forces are minimized by performing the scanning motion without tip–sample contact. We find no visible damage on DNA samples and the nominal height of 2 nm of the molecules is achieved when imaging applying a maximum normal force of ∼150 pN. In addition to topographic images, adhesion maps of DNA are simultaneously recorded showing that the minimum adhesion force occurs on top of the DNA molecules.

Comparative analysis of chloroplast DNA in Pyrus species: physical map and gene localization

A physical map of chloroplast DNA (cpDNA) of pear [Pyrus ussuriensis var. hondoensis (Nakai et Kikuchi) Rehder] was constructed using five restriction enzymes, SalI, XhoI, BamHI, SacI and PstI. This information will make it possible to investigate the phylogenetic relationships between Pyrus species. Pear cpDNA was found to be a circular molecule with a total size of about 156 kb in which two inverted repeats of 24.8 kb divide the molecule into small (17 kb) and large (90 kb) single-copy regions. The endonuclease recognition sites in the physical map were determined by single and double digestion of 13 lambda phage clones which covered the entire sequence of the pear cpDNA. Twenty nine genes were localized on the physical map of the pear cpDNA. The structure of pear cpDNA was almost the same in terms of genome size and gene order as that of tobacco cpDNA. RFLP analysis was carried out on cpDNAs from five Pyrus species (Pyrus pyrifolia, Pyrus ussuriensis, Pyrus calleryana, Pyrus elaeagrifolia and Pyrus communis). Two mutations, a recognition-site mutation and a length mutation (deletion), were found only in the cpDNA of P. pyrifolia cultivars. These mutations were localized on the physical map of pear cpDNA. The number of mutations of cpDNA in Pyrus species are small in comparison with those of other angiosperms, suggesting a high degree of genome conservatism in Pyrus species.

Genome mapping and gene analysis of Antheraea pernyi nucleopolyhedrovirus for improvement of baculovirus expression vector system

We have constructed a genome DNA map of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) and used it to identify target genes for deletion in order to improve the newly developed baculovirus expression vector system. Initially, 50 independent PstI fragments of viral DNA were obtained by shotgun cloning, and both termini of each cloned fragment were sequenced. Then, the sequence data were used for homology search against both nucleotide and amino acid sequences of other NPVs in databases. This homology search allowed us to construct a nearly complete restriction map of a viral DNA with several assumed gaps. Four additional PstI fragments covering the gaps were obtained by PCR amplification, and a complete map of a circular viral DNA, which consisted of 54 PstI fragments, was constructed. The map indicated that the AnpeNPV genome is approximately 130.2 kbp in size and possesses high similarity to the Orgyia pseudotsugata multicapsid NPV (OpMNPV) genome in both sequence and arrangement of genes. Utilizing the genome-wide high similarity between AnpeNPV and OpMNPV, we identified two target genes on the map, namely, cathepsin and chitinase genes, whose products have been proved to be involved in the degradation of recombinant proteins and the liquefaction of virus-infected insect tissues. Comparative sequence analysis of the map also revealed the lack of certain OpMNPV open reading frame (ORF) homologs and the presence of ORFs, whose homologs do not exist in OpMNPV but in other group I NPVs, providing an insight into the position of AnpeNPV in the baculovirus phylogeny.

Genome Mapping and Gene Analysis of Antheraea pernyi Nucleopolyhedrovirus for Improvement of Baculovirus Expression Vector System

We have constructed a genome DNA map of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) and used it to identify target genes for deletion in order to improve the newly developed baculovirus expression vector system. Initially, 50 independent PstI fragments of viral DNA were obtained by shotgun cloning, and both termini of each cloned fragment were sequenced. Then, the sequence data were used for homology search against both nucleotide and amino acid sequences of other NPVs in databases. This homology search allowed us to construct a nearly complete restriction map of a viral DNA with several assumed gaps. Four additional PstI fragments covering the gaps were obtained by PCR amplification, and a complete map of a circular viral DNA, which consisted of 54 PstI fragments, was constructed. The map indicated that the AnpeNPV genome is approximately 130.2 kbp in size and possesses high similarity to the Orgyia pseudotsugata multicapsid NPV (OpMNPV) genome in both sequence and arrangement of genes. Utilizing the genome-wide high similarity between AnpeNPV and OpMNPV, we identified two target genes on the map, namely, cathepsin and chitinase genes, whose products have been proved to be involved in the degradation of recombinant proteins and the liquefaction of virus-infected insect tissues. Comparative sequence analysis of the map also revealed the lack of certain OpMNPV open reading frame (ORF) homologs and the presence of ORFs, whose homologs do not exist in OpMNPV but in other group I NPVs, providing an insight into the position of AnpeNPV in the baculovirus phylogeny.

Promoter-specific or all wRAPped up in the genome?

A question that has puzzled biologists is ‘what determines where, when and how regulatory proteins bind to the genome?’ These DNA-binding proteins control the expression of specific genes and are of fundamental importance for eukaryotic cell growth and survival. In Saccharomyces cerevisiae, repressor activator protein 1 (Rap1) is one such protein, which, together with the accessory silencing proteins Sir2, Sir3 and Sir4, functions to regulate telomere length and mating. Independent of Sir proteins, Rap1 is an essential sequence-specific transcriptional regulator of many crucial S. cerevisiae genes, acting either directly as a transcriptional activator or as an accessory for binding by other regulatory proteins. By examining the specificity of Rap1–DNA binding across the entire yeast genome, Lieb et al. 1xSee all References1 now provide some interesting answers to this fundamental biological question.Yeast chromatin fragments enriched in stabilized Rap1 and Sir protein complexes were hybridized to DNA microarrays containing 12 943 DNA segments that represented every coding and intergenic region of the yeast genome. This novel approach enabled Lieb and colleagues to construct a genome-wide map of DNA–protein interactions. Careful analysis of the data identified the specific genomic targets of Rap1 and the Sir proteins. Consistent with the ability of these regulatory proteins to bind to telomeres, telomeric DNA was enriched in the Rap1 protein complexes.The analysis also allowed the identification of genes that Rap1 might regulate. To their delight, Lieb et al. found that Rap1 bound to the intergenic region immediately upstream of 362 genes. The protein products of these genes include ribosomal proteins, a continuous enzymatic pathway in glycolysis and others involved in protein synthesis. However, the most exciting finding was that, although the binding sequence for Rap1 occurs throughout the genome, Rap1 binds specifically to intergenic sequences containing potential promoters, rather than to coding sequences. This result raises the possibility that such a phenomenon might be a general property of proteins that function as transcriptional regulators but recognize DNA motifs found throughout the genome. There is no question that such a finding suggests the existence of a genome-wide mechanism that distinguishes between the potential DNA-binding sites that occur within coding regions and those in intergenic regions. The race is now on to understand how these promoter regions are ‘marked’. In the meantime, we eagerly await the results of the next study to use this powerful genomic mapping approach to identify the in vivo binding sites of other transcription factors with unknown DNA-binding specificities.

Transcript mapping and processing of mitochondrial RNA in Dictyostelium discoideum.

The circular mitochondrial genome of Dictyostelium discoideum has a size of 55,564 base pairs. We present here a complete and detailed transcription map of the mitochondrial DNA. Eight major, polycistronic transcripts encoding polypeptides, ribosomal RNAs and interspersed transfer RNAs were identified in Northern hybridization studies. Most of these polycistronic transcripts are subsequently processed into smaller mono-, di- or tricistronic RNAs. In some cases, the maturation involves endonucleolytic cleavage of the transcripts using transfer RNAs as excision signals. Primer extension experiments mapped the 5' ends of the transcripts, which may represent transcription initiation sites. Two of the polycistronic transcripts were found to be overlapping. Based on sequence alignments of the potential transcription start sites, a short oligonucleotide consensus initiation sequence has been identified which does not reveal any significant sequence homologies to known promoter regions from other organisms.

Local matching of random restriction maps

Optical mapping is a new technique to generate restriction maps of DNA easily and quickly. DNA restriction maps can be aligned by comparing corresponding restriction fragment lengths. To relate, organize, and analyse these maps it is necessary to rapidly compare maps. The issue of the statistical significance of approximately matching maps then becomes central, as in BLAST with sequence scoring. In this paper, we study the approximation to the distribution of counts of matched regions of specified length when comparing two DNA restriction maps. Distributional results are given to enable us to computep-values and hence to determine whether or not the two restriction maps are related. The key tool used is the Chen-Stein method of Poisson approximation. Certain open problems are described.

Local matching of random restriction maps

Optical mapping is a new technique to generate restriction maps of DNA easily and quickly. DNA restriction maps can be aligned by comparing corresponding restriction fragment lengths. To relate, organize, and analyse these maps it is necessary to rapidly compare maps. The issue of the statistical significance of approximately matching maps then becomes central, as in BLAST with sequence scoring. In this paper, we study the approximation to the distribution of counts of matched regions of specified length when comparing two DNA restriction maps. Distributional results are given to enable us to computep-values and hence to determine whether or not the two restriction maps are related. The key tool used is the Chen-Stein method of Poisson approximation. Certain open problems are described.

Quantitation and structural analysis of SV40 RNAs.

The 5243-bp genome of SV40 contains two transcriptional units (see Fig. 1); the early one, which is first expressed early in the lytic cycle of infection, and the late unit, which is expressed at a significant level only after the onset of viral DNA replication (). The early genes encode the viral regulatory proteins, small, and large T-antigens. These proteins play roles in replication of the viral genome and transcriptional transactivation of the late genes (see refs. , , and references therein). The late genes encode the capsid proteins that encapsulate the viral DNA into new virions. The cis-acting transcriptional elements that regulate the synthesis of both the early and late viral RNAs (see refs. , and references therein), the mechanisms that regulate the temporal expression of the SV40 genome (see ref. and references therein) and the patterns by which the viral transcripts are spliced (see refs. , and references therein) are well understood. In this chapter, we describe methods for quantifying and mapping the 5 ends of the viral RNAs synthesized in SV40-transfected cells. Minor variations of these methods can be used to map the 3 ends as well () and the splice sites () of the SV40 RNAs synthesized, not only in SV40-transfected cells, but also in SV40-infected and transformed cells. Open image in new window Fig. 1. Schematic of the SV40 genome. (A) Map of wild-type SV40 DNA. (B) Schematic of the origin-promoter region of SV40. This bidirectional promoter controls both early and late transcription. The thick and thin arrows indicate the positions at which transcription initiates and the direction transcription proceeds. The objects indicate the cis-acting sequences and the transcription factors that bind in trans. The nucleotide numbering system is that of Buchman et al. ().

Bacteriophage phiYeO3-12, specific for Yersinia enterocolitica serotype O:3, is related to coliphages T3 and T7.

Bacteriophage phiYeO3-12 is a lytic phage of Yersinia enterocolitica serotype O:3. The phage receptor is the lipopolysaccharide O chain of this serotype that consists of the rare sugar 6-deoxy-L-altropyranose. A one-step growth curve of phiYeO3-12 revealed eclipse and latent periods of 15 and 25 min, respectively, with a burst size of about 120 PFU per infected cell. In electron microscopy phiYeO3-12 virions showed pentagonal outlines, indicating their icosahedral nature. The phage capsid was shown to be composed of at least 10 structural proteins, of which a protein of 43 kDa was predominant. N-terminal sequences of three structural proteins were determined, two of them showing strong homology to structural proteins of coliphages T3 and T7. The phage genome was found to consist of a double-stranded DNA molecule of 40 kb without cohesive ends. A physical map of the phage DNA was constructed using five restriction enzymes. The phage infection could be effectively neutralized using serum from a rabbit immunized with whole phiYeO3-12 particles. The antiserum also neutralized T3 infection, although not as efficiently as that of phiYeO3-12. phiYeO3-12 was found to share, in addition to the N-terminal sequence homology, several common features with T3, including morphology and nonsubjectibility to F exclusion. The evidence conclusively indicated that phiYeO3-12 is the first close relative of phage T3 to be described.

Physical map of a Plutella xylostella granulovirus genome.

A plasmid library of restriction DNA fragments of a Plutella xylostella granulovirus (PxGV) was constructed, which covered all 9 EcoRI and all 19 HindIII fragments, and 18 of 20 PstI fragments. Restriction endonuclease analysis and DNA-DNA hybridization analysis of the cloned viral DNA fragments in the library and the restricted viral DNA fragments were carried out for construction of a physical map of PxGV DNA for ApaI, BamHI, BglII, EcoRI, HindIII, KpnI, PstI, SacII, XbaI and XhoI. The size of PxGV genome was estimated to be 102.2 kb. Location of the left-most DNA fragment on the physical map that contained granulin gene was determined by hybridization using a DNA probe of granulin gene of Trichoplusia ni granulovirus. Four homologous regions interspersed on the genome were also determined by DNA-DNA hybridization analysis.

Isolation and analysis of cDNA within a 300 kb Arabidopsis thaliana genomic region located around the 100 map unit of chromosome 1.

In order to analyze the organization of genes located at the 100 map unit of chromosome 1, we screened cDNAs hybridized with approximately 300kb contiguous DNA using four P1 clones and one YAC clone. A total of 40 kinds of cDNA were isolated, and their entire sequences were determined. A comparison with the GenBank/EMBL database indicated that three of the cDNAs have been found in Arabidopsis, and that similar sequences to 18 of the cDNAs had been detected in Arabidopsis or other organisms. cDNAs were aligned on a physical map of the contiguous DNA, and the transcriptional direction of each cDNA was determined. This contiguous DNA contains a large direct repeat, which contains five genes. In addition, identical or very similar sequences to two cDNAs are located in a narrow region. Thus, a total of 50 genes were identified, and the gene density was revealed to be approximately one gene every 6kb. In addition, cDNA sequencing revealed the existence of unusual transcripts. A sequence of seven cDNAs seemed to have no significant open reading frames. Furthermore, the existence of antisense RNA and the possibility of alternative splicing were also revealed.

Optical PCR: genomic analysis by long-range PCR and optical mapping.

Optical mapping is an approach for the rapid, automated, non-electrophoretic construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genomic loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors.

Alterations in organization and transcription of the mitochondrial genome of cytoplasmic male sterile sugar beet (Beta vulgaris L.).

We have constructed a physical map of the mitochondrial DNA of a cytoplasmic male sterile (CMS) sugar beet line, TK81-MS, and compared it with that published for normal fertile sugar beet (cv. TK81-O) to clarify the differences between the CMS and normal mitochondrial genomes. The TK81-MS genome is present as a single circular molecule of 481.8 kb, or as two molecules of 184.9 and 296.9 kb. The CMS genome was found to be highly rearranged relative to the normal mitochondrial genome, with at least fifteen rearrangement and/or inversion events being required to align the two DNAs. Analysis of transcription patterns of known mitochondrial genes and rearranged regions revealed six genes, coxI, coxII, atpA, atp6, rps3, and orf324, whose expression is altered in the CMS line relative to the normal line. Of these six, only the coxI transcript pattern differs between male-sterile and fertility-restored genotypes, making it likely that the coxI locus is involved in mediating CMS in sugar beet.

GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and encodes a protein with several possible membrane-spanning domains.

Flowering of Arabidopsis is promoted by long days and delayed by short days. Mutations in the GIGANTEA (GI) gene delay flowering under long days but have little or no effect under short days. We have now isolated the GI gene and show that it encodes a novel, putative membrane protein. By comparing the sequence of the Arabidopsis gene with that of a likely rice orthologue and by sequencing mutant alleles, we identify regions of the GI protein that are likely to be important for its function. We show that GI expression is regulated by the circadian clock with a peak in transcript levels 8-10 h after dawn. The timing, height and duration of this peak are influenced by daylength. We analysed the interactions between GI and the LHY, CCA1 and ELF3 genes, previously shown to affect daylength responses; we show that the rhythmic pattern of GI expression is altered in the elf3, CCA1-OX and lhy genotypes, and that CCA1 and LHY expression are reduced by gi mutations. Our results are consistent with the idea that GI plays an important role in regulating the expression of flowering time genes during the promotion of flowering by photoperiod.

Structure Maps of Close-Packed Layered Compounds and DNA

Close-packed layered compounds such as FexGa2S3+x and NxC3Al4+x (x=0–4) are analyzed as chains of covalent-bonded S=Ga(–S–Fe)x–S–Ga=S or Al≡C(–Al=N)x–Al=C=Al (–N=Al)x″–C≡Al with delocalized bonds. The rules for valence compounds allow RM, R(MR)n (n=1,2,3), RMMR, and different combinations and substitutions. The conformation of the chains is characterized by the Jagodzinski–Wyckoff symbols c and h and a similar notation s and t at the occupation of tetrahedral sites. The interactions between the different atoms are analyzed by structure maps with the self-coordination numbers of nearest and next-nearest neighbors as parameters. A similar analysis of 18 different DNA or RNA reveals four different groups within different fields of the structure map similar to solid solutions or nonperiodic polytypes of layered compounds.