Chromosomal DNA Bands Research Articles

The Effect of Coumarin Derivatives(compounds) on the Vibrio cholerae Isolates from Different Clinical Iraqi Sources

From a large number of bacterial samples collected from different hospital in Iraq in central health laboratory ,only ten isolates were identified primary as Vibrio. A number of morphology and biochemical test were carried out to complete this identification that showed all bacterial isolates were related to Vibrio cholerae .In this study all Vibrio isolates were investigated for Bio typing and the result showed that all (10) isolate were related to (Eltor biotypes) .Also, the susceptibility test towards eight antibiotics were carried out . Results shows that ciprofloxacin , Norfloxacin, Erythromycin, Ampicillin, ceftriaxone and Amikacin were the most effective antibiotics and their resistance percentage were 20%,20%,20, 20,30% and 30% respectively ,While Chloramphenicol and Co- trimoxazole were less effective and their resistance percentage were 90% both of them. Three (3,5,6) isolates V. cholerae were selected depending on results of antibiotics sensitivity tests as showed multiple –antibiotics resistance(100%). Then tested to study the effect of coumarin derivatives compounds (1, 2, 3 ) which showed inhibitory effect on V. cholera (3,5,6) isolates and the compound ( 3 ) showed the highest antibacterial activity of (12,15,14 mm) of inhibition zone diameter against V. cholera (3,5,6) isolates respectively. Also, these Iraqi isolates (3,5,6) used to test the effect of acridine orange (0.1%) as acuring agent , the results showed that all (3) isolates V. cholerae were sensitive to (ciprofloxacin, ceftrixone and Norfloxacin), While the rest were resistance to remained five antibiotics. The results of Agarose –gel electrophoresis of both normal V. cholerae (3,5,6) and cured isolates showed the presence of chromosomal and plasmid DNA bands in the normal case ,While only chromosomal DNA bands occur with V. cholerae (isolate 8) treated with an acridine orange at concentration of (10-2 to 10-4).

The Effect of Coumarin Derivatives(compounds) on the Vibrio cholerae Isolates from Different Clinical Iraqi Sources

From a large number of bacterial samples collected from different hospital in Iraq in central health laboratory ,only ten isolates were identified primary as Vibrio. A number of morphology and biochemical test were carried out to complete this identification that showed all bacterial isolates were related to Vibrio cholerae .In this study all Vibrio isolates were investigated for Bio typing and the result showed that all (10) isolate were related to (Eltor biotypes) .Also, the susceptibility test towards eight antibiotics were carried out .
 Results shows that ciprofloxacin , Norfloxacin, Erythromycin, Ampicillin, ceftriaxone and Amikacin were the most effective antibiotics and their resistance percentage were 20%,20%,20, 20,30% and 30% respectively ,While Chloramphenicol and Co- trimoxazole were less effective and their resistance percentage were 90% both of them. Three (3,5,6) isolates V. cholerae were selected depending on results of antibiotics sensitivity tests as showed multiple –antibiotics resistance(100%). 
 Then tested to study the effect of coumarin derivatives compounds (1, 2, 3 ) which showed inhibitory effect on V. cholera (3,5,6) isolates and the compound ( 3 ) showed the highest antibacterial activity of (12,15,14 mm) of inhibition zone diameter against V. cholera (3,5,6) isolates respectively.
 Also, these Iraqi isolates (3,5,6) used to test the effect of acridine orange (0.1%) as acuring agent , the results showed that all (3) isolates V. cholerae were sensitive to (ciprofloxacin, ceftrixone and Norfloxacin), While the rest were resistance to remained five antibiotics.
 The results of Agarose –gel electrophoresis of both normal V. cholerae (3,5,6) and cured isolates showed the presence of chromosomal and plasmid DNA bands in the normal case ,While only chromosomal DNA bands occur with V. cholerae (isolate 8) treated with an acridine orange at concentration of (10-2 to 10-4).

A reliable method for sexing unincubated bird eggs for studying primary sex ratio

In birds, offspring sex ratio manipulation by mothers is now well established with potentially important consequences for evolution and animal breeding. In most studies on primary sex ratio of birds, eggs are sexed after incubation by the use of PCR methods targeted to the sex-linked CHD1 genes. Sexing of unincubated eggs would be preferred, but as fertile and infertile blastodiscs cannot be distinguished macroscopically, errors could arise from PCR amplifications of parental DNA associated with the vitelline membrane of infertile eggs. In this study, we stained blastodiscs without the vitelline membrane with Hoechst 33342. This allowed unequivocal distinction between fertile and infertile blastodiscs. Fertile blastodiscs contained thousands of fluorescent nuclei, whereas no nuclei were seen in infertile eggs. In addition, after nucleic acid analysis, fertile blastodiscs yielded much stronger chromosomal DNA and CHD1-targeted PCR bands on agarose gels compared with infertile blastodiscs. These findings indicate that fertile blastodiscs contain much more embryonic DNA than parental DNA, allowing reliable sexing of the fertile eggs. The differences between fertile and infertile blastodiscs in chromosomal DNA and CHD1 PCR banding intensities alone could also be used to distinguish fertile from infertile eggs without using Hoechst staining. We conclude that identifying fertile blastodiscs either by Hoechst staining or by analyzing the yield of chromosomal DNA and CHD1-PCR products, combined with CHD1-targeted PCR amplification, presents an easy and reliable method to sex unincubated eggs.

Cytological and electrophoretic karyotyping of the chestnut blight fungus Cryphonectria parasitica

The karyotypes of nine strains including three transformants of the chestnut blight fungus Cryphonectria parasitica were analyzed by pulsed-field gel electrophoresis (PFGE) and cytology using a fluorescence microscope. Cytology of the mitotic metaphase showed n = 9 for both standard strain EP155 and field strain GH2 infected by Cryphonectria hypovirus 3. Chromosomes were morphologically characterized by size, heterochromatic segment, and constriction. PFGE resolved 5 or 6 chromosomal DNA bands ranging from 3.3 Mbp to 9.7 Mbp, but accurate determination of the chromosome number was hampered by clumping of some bands. Banding profiles in PFGE were similar among the strains except for GH2, in which a chromosome translocation was detected by Southern blot analysis. By integrating the data from cytology and PFGE, the genome size of C. parasitica was estimated to be ca. 50 Mbp. This is the first report of a cytological karyotype in the order Diaporthales.

Electrophoretic karyotype of Flammulina velutipes and its variation among monokaryotic progenies

The karyotype of Flammulina velutipes (Curt. : Fr.) Sing. was investigated using contour-clamped homogeneous electric fields (CHEF) gel electrophoresis. A parental dikaryotic stock, JA, was resolved into at least eight chromosomal DNA bands ranging from 1.4- to 4.9-megabase (Mb) pairs. Overall, little size variation was found among monokaryotic strains with a few major exceptions. Among 13 monokaryotic progenies examined, 11 strains were resolved into at least eight chromosomal DNA bands in a manner similar to the parent dikaryon, whereas the other 2 were resolved into at least seven chromosomes lacking the 2.1-Mb chromosome possessed in the former. A slightly larger size variation was found in a chromosome carrying ribosomal DNA. An estimated haploid genome size of this stock was 24.0Mb or more.

Extensive chromosome translocation in a clinical isolate showing the distinctive carbohydrate assimilation profile from a candidiasis patient

Variation of the electrophoretic karyotype is common among clinical strains of Candida albicans and chromosome translocation is considered one of the causes of karyotypic variation. Such chromosome translocations may be a mechanism to confer phenotypic diversity on the imperfect fungus C. albicans. A clinical strain, TCH23, from a vaginal candidiasis patient shows distinct carbohydrate assimilation profile, serotype B, no chlamydospore formation and an atypical karyotype (Asakura et al., 1991). To examine the taxonomic relationship among C. albicans, Candida dubliniensis and this strain, we sequenced the internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA. The ITS1 sequence of TCH23 was identical with that of C. albicans but not of C. dubliniensis. Thus, strain TCH23 was classified as a variant of C. albicans with an atypical phenotype. The chromosomal DNAs of this strain were resolved into 13 bands on pulse-field gel electrophoresis (PFGE). Using DNA probes located at or near both ends of each chromosome of C. albicans, we investigated the chromosome organization of this strain. Referring to the SfiI map of C. albicans 1006 (Chu et al., 1993), we found that seven chromosomal DNA bands in strain TCH23 were reciprocal chromosome translocations. One homologue from chromosomes 1, 2 and 6 and both homologues from chromosomes 4 and 7 participated in these events. One translocation product was composed of three SfiI fragments, one each from chromosomes 2, 4 and 7. We deduced the breakpoints of chromosome translocation from the physical map of this strain; between 1J and 1J1, between 2A and 2U, both ends of 4F2, between 6C and 6O and both ends of 7F.

Karyotype analysis of interspecific fusants of basidiomycetes by pulsed-field gel electrophoresis

The purpose of this research was to analyze the karyotype of the interspecific fusants of twoPleurotus species. Auxotrophic mutants derived from the cultivated strain ofP. ostreatus andP. cornucopiae were used. Protoplasts were fused electrically, and the fusants were selected under auxotrophic complementation. Esterase isozyme analysis showed that several fusants had isozyme bands originating from both parental strains, and others had unilateral isozyme bands. The fusant that had expressed isozyme bands of both parental strains showed chromosomal DNA bands of both of the parental strains in pulsedfield gel electrophoresis analysis. Despite the above results, the chromosomal composition of the fusants obtained by the pulsed-field gel electrophoresis did not exhibit all of the bands of both fusion parents.

Inter- and intra-species karyotype variations among microsporidia of the genus Encephalitozoon as determined by pulsed-field gel electrophoresis.

Disseminated infections due to microsporidia of the genus Encephalitozoon are detected increasingly, especially in patients with AIDS. Identification of microsporidia can be achieved by a variety of immunological and molecular methods. This study evaluates the feasibility of pulsed-field gel electrophoresis (PFGE) for the analysis of karyotypes of the 3 known species of this genus (Encephalitozoon cuniculi, Encephalitozoon hellem and Encephalitozoon intestinalis) and of 2 of the 3 known E. cuniculi strains (strains I and III). Eleven chromosomal DNA bands were resolved for E. cuniculi and 10 chromosomal DNA bands for E. hellem and E. intestinalis, with molecular sizes ranging from 231 to 320 kb, from 197 to 288 kb and from 195 to 285 kb, respectively, resulting in estimated genome sizes of about 3.0 Mb, 2.5 Mb and 2.4 Mb. Different PFGE chromosomal banding patterns indicate that not only E. cuniculi, as previously described, but also E. hellem, represent a heterogeneous entity. PFGE is a valuable method of evaluating inter- and intra-species variations among Encephalitozoon species that may enable the identification of environmental sources of infection and modes of transmission.

Separation of Candida fermentati comb. nov. from Candida guilliermondii by DNA base composition and electrophoretic karyotyping

Summary DNA base composition and electrophoretic karyotypes of eight strains of Candida guilliermondii (Castellani) Langeron et Guerra including the type strains of the species and its synonym Torula fermentati Saito were determined by using thermal denaturation profiles (Tm) and the CHEF (contour-clamped homogeneous electric field) system of pulsed field gel electrophoresis (PFGE), respectively. These strains are separated into two distinct groups by the data obtained. The four strains in group I, including the type strain of C. guilliermondii , have 44.1–44.6 mol% G+C and homogeneous electrophoretic karyotypes consisted of 6–8 chromosomal DNA bands ranging in size from 0.4 to 2.1 Mb, while the other four strains in group II, including the type strain of T. fermentati , have 49.4–49.8 mol% G+C and almost identical karyotypes composed of 6 bands with molecular sizes ranging from 0.9 to 1.8 Mb. The approximately 5.0% difference in G+C contents and completely different electrophoretic karyotypes between the two groups demonstrate that these two groups represent two separate species. As a result, a new combination Candida fermentati (Saito) Bai comb. nov. is proposed.

Electrophoretic karyotype of Mucor circinelloides

Contour-clamped homogeneous electric field (CHEF) gel electrophoresis was used to separate chromosomal size DNA molecules of two Mucor circinelloides strains. Electrophoretic karyotypes revealed the presence of eight distinct bands for the M. circinelloides f. lusitanicus strain, and four, presumably multiple, bands for the M. circinelloides f. gryseo-cyanus strain. The approximate sizes of the resolved chromosomal DNA bands ranged from 2.3 to 8.1 Mb, giving estimated genome sizes of 38.7 and 32.6 Mb, respectively. Hybridisation techniques were used to assign the leuA gene to a chromosome.

Assignment of genes to pulse-field separated chromosomes of Schizophyllum commune

Chromosomal DNAs of the basidiomycete Schizophyllum commune were separated by Contour-Clamped Homogeneous Electric Field Electrophoresis (CHEF). The estimated sizes of the chromosomal DNAs ranged from 4·7 Megabase pairs (Mbp) to 1·6 Mbp, totalling 35·6 Mbp. Using sequences from 20 cloned genes we obtained hybridization signals to at least 8 out of 11 chromosomal DNAs. The mating-type genes A and B were mapped to the largest and the smallest chromosomal DNA band, respectively. Developmentally regulated genes which are under the control of the mating-type genes, were localized to five different chromosomal DNA bands.

Electrophoretic karyotype and gene assignment to chromosomes of Aspergillus oryzae.

An electrophoretic karyotype of Aspergillus oryzae was obtained by transverse altering-field electrophoresis. Seven chromosomal bands were found. With Schizosaccharomyces pombe chromosomes as size standards, we estimated the sizes of the chromosomes to be 7.0, 5.2, 5.0, 4.5, 4.0, 3.7, and 2.8 megabase pairs (Mbp). The chromosomal DNA bands were identified with use of 13 cloned genes including ribosomal DNA. The intensity of ethidium staining and the results of Southern blotting with 100 random clones isolated from A. oryzae suggested that the smallest band migrated as doublet and that the total genome size was approximately 35 Mbp.

A small chromosome of Leishmania (Viannia) braziliensis contains multicopy sequences which are complex specific

Orthogonal Field Alternating Gel Electrophoresis (OFAGE) has been used to show a band of approximately 260 kb which is stained intensely with ethidium bromide in Leishmania (V.) braziliensis stock M2903. This small chromosome (sc-2903), as well as a 50 kb and a 200 kb chromosome seen in L. (L.) mexicana and L. (L.) amazonensis, respectively, are stably maintained and linear. When used as a hybridisation probe, sc-2903 showed homology to large chromosomal DNA bands and to a multiplicity of genomic fragments in all braziliensis stocks tested, indicating either different sequences, different copy numbers or both but no hybridisation to mexicana stocks. It is possible that these sequences are present in all members of the braziliensis complex and are not related to LD1 or any other previously published small chromosome sequences. However, at least one clone isolated from a sc-2903 library recognised genomic DNA of stocks belonging to the braziliensis, mexicana and donovani complexes. Our results suggest that the clone carries sequence(s) that are repeated and shared between stocks of different complexes but with a variable genomic distribution.

Major chromosomal length polymorphisms are evident after meiosis in the phytopathogenic fungus Leptosphaeria maculans.

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size.

Electrophoretic karyotypes of the elm tree pathogen Ophiostoma ulmi (sensu lato)

Pulsed field gel electrophoresis using OFAGE, TAFE, and CHEF systems has been used to more fully characterize karyotypic variation within the two closely related fungal species of Ophiostoma ulmi sensu lato. Twelve wild-type and laboratory strains, representing the less aggressive species O. ulmi and both of the biotypes of the more aggressive species O. novo-ulmi were studied and their karyotypes determined. Depending on the strain, a minimum of four to a minimum of eight chromosomal DNA bands were present that fall into three distinct size classes, with one exception. Strain CESS16K (O. novo-ulmi, North American aggressive subgroup) contains a unique chromosomal DNA band which comigrated near a Saccharomyces cerevisiae chromosome of 0.95 Mb. This unique band was the smallest O. ulmi s. l. chromosomal DNA observed. Seven of the twelve strains shared a common chromosomal DNA banding pattern, whereas each of the other five had a unique karyotype. There was no correlation between chromosome profile and species, as some O. novo-ulmi and O. ulmi strains shared common electrophoretic karyotypes.

Electrophoretic karyotype and gene assignment to resolved chromosomes of Trichoderma spp.

A molecular karyotype for three different Trichoderma species (T. harzianum, T. viride, and T. reesei) was determined by using two different systems: countour-clamped electric-field and rotating-electrode electrophoresis. Six chromosomal DNA bands were observed in T. harzianum and T. reesei and five in T. viride. The sizes of these molecules were estimated by their mobility relative to the Schizosaccharomyces pombe chromosomes and ranged between 2.2 and 7.4 megabase pairs (mbp). The estimated genome sizes range from 31 to 39 mbp. A number of genes were located in the different chromosomes by means of Southern analysis. The implications of these findings are discussed.

Electrophoretic karyotyping of wild-type and mutant Trichoderma longibrachiatum (reesei) strains.

An electrophoretic karyotype of Trichoderma longibrachiatum (reesei) was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Seven chromosomal DNA bands were separated in the wild-type T. longibrachiatum strain QM6a. The sizes of the chromosomal DNA bands ranged from 2.8 to 6.9 Mb, giving an estimated total genome size of about 33 Mb. The electrophoretic karyotype of the strain QM6a was compared to three hyper-celluloytic mutant strains, QM9414, RutC30 and VTT-D-79125. The chromosome pattern of the mutant QM9414 was quite similar to that of the wild-type QM6a except that the smallest chromosome differed somewhat in size. The VTT-D-79125 and RutC30 strains, which have undergone several mutagenesis steps, showed striking differences in their karyotype compared to the initial parent. The chromosomal DNA bands were identified using the previously characterized T. longibrachiatum genes (egl1, egl2, cbh1, cbh2, pgk1, rDNA) and random clones isolated from a genomic library. In all strains the cellulase genes cbh1, cbh2 and egl2 were located in the same linkage group (chromosome II in the wild-type), while the main endoglucanase, egl1, hybridized to another chromosomal DNA band (chromosome VI in the wild-type).

Molecular karyotype and chromosomal localization of genes encoding two major surface glycoproteins, gp63 and gp46/M2, hsp70, and β-tubulin in cloned strains of several Leishmania species

The molecular karyotypes of several Leishmania isolates ( Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania panamensis, Leishmania donovani, Leishmania major, Leishmania aethiopica, Leishmania tropica, Leishmania enriettii) have been analyzed by clamped homogeneous electric field (CHEF) gel electrophoresis. The chromosomal localization of genes encoding 2 major surface glycoproteins, gp63 and gp46/M2, heat shock protein 70 (hsp70), and β-tubulin was determined for cloned isolates of 8 of these Leishmania species. The chromosome size class assignment of hsp70 genes was most conserved in that all species contained a single hybridizing DNA band of approximately 1200 kb. The β-tubulin gene probe hybridized predominantly to large (1600–1750 kb) chromosome-size DNA and to 1–5 additional bands, the number of which depended on the species. The number and size of DNA bands hybridizing to gp63 or gp46/M2 gene probes were not uniformly conserved among species. In contrast to previous reports of gp63 genes being located on a single chromosome, using various CHEF gel conditions we observed a Leishmania major gp63 gene probe hybridizing to at least 2 chromosomal DNA bands in the New World species and in L. tropica. Gp46/M2 genes were located on 1 band in L. donovani, L. major, and L. aethiopica or 2 bands in L. tropica and L. amazonensis, but surprisingly, do not hybridize to any chromosomal DNA of species in the L. braziliensis complex or in L. enriettii. Whenever both genes were present in a species, gp63 and gp46/M2 genes were located on different chromosomal DNA bands.

Pulsed-field gel electrophoretic analysis of Schizophyllum commune chromosomal DNA

Six chromosomal DNA bands of Schizophyllum commune have been resolved using transverse alternating field electrophoresis. The estimated sizes of the chromosomal DNAs ranged from 5.1 to 1.2 megabase pairs (Mb), the total genome size being approximately 35-36 Mb. Chromosomal length polymorphisms were found between the two S. commune isolates examined. The DNA bands corresponding to the two chromosomes containing the A and B mating-type loci were identified in hybridization experiments using probes specific to their respective linkage groups. The utility of eluted chromosomal DNAs as hybridization probes to select clones from genomic libraries, and the use of these clones in transformation experiments to identify genes of interest, are discussed.

Colicins E7 and E8 degrade DNA in sensitive bacteria

The primary target of colicin E7 in sensitive bacteria are their DNA molecules. In agarose gel electrophoresis of lysates of cells treated with colicin E7, both chromosomal and plasmid DNA bands disappear, in direct relation to E7 concentration and to the duration of treatment. DNA degradation is followed by a cessation of DNA synthesis. In E7-immune bacteria, no damage to DNA due to colicin E7 occurs. The mode of action of colicin E7 thus appears to be equal to that of colicin E2. Also, colicin E8 causes a distinct damage to chromosomal and plasmid DNA in sensitive, but not in immune bacteria. None of the colicins E1, E3, E4, E5, E6 or E9 has any influence on bacterial DNA.