The complete karyotype of Aspergilius niger: the use of introduced electrophoretic mobility variation of chromosomes for gene assignment studies

A method is described for unambiguous assignment of cloned genes to Aspergillus niger chromosomes by CHEF gel electrophoresis and Southern analysis. All of the eight linkage groups (LGs), with the exception of LG VII, have previously been assigned to specific chromosomal bands in the electrophoretic karyotype of A. niger. Using a LG VII-specific probe (nicB gene of A. niger) we have shown that LG VII corresponds to a chromosome of about 4.1 Mb. Furthermore, genetic localization of three unassigned genes (glaA, agIA and pepA) in strains in which these genes had been replaced by a selectable marker gene led to a revised karyotype for the chromosomes corresponding to LGs VIII and VI. The revised electrophoretic karyotype reveals only 5 distinct bands. The presence of three pairs of equally sized chromosomes precluded assignment of genes to one specific chromosome in the wild-type strain. However, unambiguous chromosome assignment of cloned genes using CHEF-Southern analysis was demonstrated using a set of A. niger strains with introduced chromosomal size variation. The availability of these tester strains obviates the need to isolate or construct mutant. strains for the purpose of chromosome assignment.     

An electrophoretic karyotype and assignment of ribosomal genes to resolved chromosomes of Pneumocystis carinii

Pulsed-field gel electrophoresis was used to generate a molecular karyotype of chromosomes from the opportunistic AIDS pathogen, Pneumocystis carinii. P. carinii cysts and trophozoites were isolated from immunosuppressed rats, lysed in situ in agarose blocks, and subjected to orthogonal-field gel electrophoresis (OFAGE) and contour-clamped homogeneous-field gel electrophoresis (CHEF). OFAGE and CHEF gels resolved, respectively, 16 or 20 chromosome bands ranging in size from 0.32-1.5 megabase pairs. Summation of the estimated sizes of these chromosomes suggested a total genome complexity for P. carinii of 8-16 megabase pairs. Homologous probes for the genes encoding the 18S, 5.8S, and 5S ribosomal RNAs were hybridized to filter blots of the pulsed-field gels to map these genes to specific P. carinii chromosomes.     

Preparation of chromosome-specific paints and complete assignment of chromosomes in the pig flow karyotype

Sorted chromosomes from each of the 20 clusters of the male porcine bivariate flow karyotype were amplified and biotinylated using DOP-PCR. The chromosomes comprising each cluster were identified by fluorescence in situ hybridization (FISH) of the 20 probes to R-banded male pig metaphase spreads. A standard flow karyotype for the pig is presented.     

Variation in the electrophoretic karyotype analysed by the assignment of DNA probes in Candida albicans

By using pulsed-field gel electrophoresis, we have separated the entire chromosome bands and examined the electrophoretic karyotypes of 27 strains of Candida albicans. The electrophoretic karyotype varied widely among these strains. Their chromosomal DNAs were resolved into 7-12 bands ranging in size from 0.42 to 3.0 Mb. Most of the separated chromosomal bands were assigned by eight cloned C. albicans DNA probes. These results suggest that the haploid number of C. albicans chromosomes is eight. Each of the probes hybridized specifically to one or two bands of similar size in most strains. With the exception of the MGL1 probe, when two bands were detected by one probe, the size of one of them was very conserved whilst the other was of fairly variable size. The sizes of the chromosome bands assigned by the MGL1 probe were much more variable. As C. albicans is considered to be a diploid organism, it is inferred that the karyotype polymorphism between strains is mainly derived from wide size heterogeneity in one of the homologous chromosomes. Furthermore, we have confirmed species-specific and strain-specific variation in medically important Candida species (C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata). Electrophoretic karyotype analysis is thus useful for species assignation. The TUB2 probe, encoding C. albicans beta-tubulin, hybridized to the chromosomal DNA of all the Candida species examined, but four C. albicans probes exhibited cross-species hybridization with C. stellatoidea only. The karyotype of C. stellatoidea seems to be within the range of the intraspecies variation observed in C. albicans.     

The Bombyx mori karyotype and the assignment of linkage groups

Lepidopteran species have a relatively high number of small holocentric chromosomes (Bombyx mori, 2n = 56). Chromosome identification has long been hampered in this group by the high number and by the absence of suitable markers like centromere position and chromosome bands. In this study, we carried out fluorescence in situ hybridization (FISH) on meiotic chromosome complements using genetically mapped B. mori bacterial artificial chromosomes (BACs) as probes. The combination of two to four either green or red fluorescence-labeled probes per chromosome allowed us to recognize unequivocally each of the 28 bivalents of the B. mori karyotype by its labeling pattern. Each chromosome was assigned one of the already established genetic linkage groups and the correct orientation in the chromosome was defined. This facilitates physical mapping of any other sequence and bears relevance for the ongoing B. mori genome projects. Two-color BAC-FISH karyotyping overcomes the problem of chromosome recognition in organisms where conventional banding techniques are not available.     

Null and electrophoretic mobility mutants in the structural gene for L-lactate dehydrogenase of Saccharomyces cerevisiae

A mutant lacking L-lactate dehydrogenase (EC 1.1.2.3) of Saccharomyces cerevisiae was isolated by its inability to grow on minimal medium with L-lactate as a carbon source. A simple activity gel assay for visualization of this enzyme and the two D-lactate dehydrogenases in this organism (EC 1.1.2.4 and 1.1.1.28) was developed. This enabled us to screen spontaneous and ethylmethanesulfonate-induced back mutants for electrophoretic mobility. Two mutants with a mobility faster than that of the wild type were isolated, and proved to be allelic to the L-lactate dehydrogenase negative mutant.     

Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: electrophoretic mobility studies

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.     

The analysis of hidden electrophoretic variation: Interspecific electrophoretic differentiation and amino acid divergence

Summary In a study of 25 human variants and 23 evolutionary alleles of hemoglobin we show that intraspecific and interspecific patterns of electrophoretic variability are not comparable. Significant deviation from the predicted electrophoretic differentiation between evolutionary alleles is normally found only when amino acid sequence divergence exceeds 10%. When two sequences had diverged at less than 30 out of 287 amino acid residues sites, only 7% of comparisons showed significant deviations from the expected difference of electrophoretic mobility, while significant deviation was shown by 57% of comparisons involving 30–40 residue differences, by 79% in the case of 51–60 differences and by all of the comparisons involving more than 60 differences. In contrast, human variants, which differ by only one or two amino acid residues (less than 1% difference), had significant deviations in 58% of comparisons. Those mutations that appear as fixed differences in the evolutionary material probably represent only a subset of the mutations which can appear within the species. The results suggest that statistical comparisons such as genetic distance may not measure the same process within a species as between species. This is due not to inherent problems with the statistic, but rather to inherent differences in the nature of molecular changes that are detectable by electrophoresis at different stages of population divergence.     

The structural gene for the ribosomal protein S18 in Escherichia coli. II. Chemical studies on the protein S18 having an altered electrophoretic mobility

A mutant of Escherichia coli strain CR341 has an altered 30 S ribosomal protein S18. The alteration involves a change in the electrophoretic mobility of S18. S18 proteins were purified from the mutant and the parent strain, respectively, and their amino acid composition and tryptic peptides were compared. The results have shown that the mutational alteration involves substitution of cysteine for arginine. In addition, we determined the electrophoretic mobility of S18 proteins modified by ethyleneimine. The modification, which involves conversion of cysteine residues to S-(2-aminoethyl)cysteine, causes a greater electrophoretic mobility increase in the mutant protein than in the wild type protein, resulting in identical mobilities for the aminoethylated proteins. This experiment gives further support to the conclusion that the original mobility difference between mutant and wild type proteins is due to the mutational substitution of cysteine for arginine. The S18 obtained from a recombinant was also studied. The recombinant protein was found to have the mobility of the wild type protein and the wild type primary structure, as judged by amino acid composition and tryptic peptide analysis. This recombinant was obtained from the mutant by introducing Hfr strain G10 chromosome segments in the region between 70 and 10 minutes, and not in the str-spc region at 64 minutes, as described in the preceding paper. These results, together with those in the preceding paper, show that the mutation studied here is in the structural gene for S18, and that it maps outside the str-spc region.     

Some properties of the chloroplast envelope as revealed by electrophoretic mobility studies of intact chloroplasts

The electrophoretic mobility of mature spinach (Spinacia oleracea L. var. Americana) chloroplasts sampled over a 7-month period was between −2.03 and −2.45 micrometers per second per volt per centimeter when suspended in a solution containing 1 millimolar CaCl₂. The surface charge density of EDTA-treated chloroplasts was calculated to be −7,400 electrostatic units per square centimeter representing, on the average, one electronic charge per 645 square Angstroms. Electrophoretic mobility increases during plastid maturation. Calcium, but not magnesium, generally stabilized the envelope of isolated plastids against small increases in surface charge that occur with time in the absence of calcium. Pronase caused a sharp, but temporary, decrease in the electrophoretic mobility of chloroplasts. This was interpreted as representing a transient binding of pronase to the envelope surface during proteolysis. No −SH groups were detected on the surface of the plastid envelope. Inasmuch as the isoelectric point of intact chloroplasts was found to be at pH 4.5, it is likely that the major part of the total surface charge results from the presence of exposed carboxyl groups of intrinsic envelope proteins that are not readily hydrolyzed by mild pronase treatment.     

"Bar-coding" primate chromosomes: molecular cytogenetic screening for the ancestral hominoid karyotype

Two recently introduced multicolor FISH approaches, cross-species color banding (also termed Rx-FISH) and multiplex FISH using painting probes derived from somatic cell hybrids retaining fragments of human chromosomes, were applied in a comparative molecular cytogenetic study of higher primates. We analyzed these "chromosome bar code" patterns to obtain an overview of chromosomal rearrangements that occurred during higher primate evolution. The objective was to reconstruct the ancestral genome organization of hominoids using the macaque as outgroup species. Approximately 160 individual and discernible molecular cytogenetic markers were assigned in these species. Resulting comparative maps allowed us to identify numerous intra-chromosomal rearrangements, to discriminate them from previous contradicting chromosome banding interpretations and to propose an ancestral karyotype for hominoids. From 25 different chromosome forms in an ancestral karyotype for all hominoids of 2N=48 we propose 21. Probes for chromosomes 2p, 4, 9 and Y were not informative in the present experiments. The orangutan karyotype was very similar to the proposed ancestral organization and conserved 19 of the 21 ancestral forms; thus most chromosomes were already present in early hominoid evolution, while African apes and human show various derived changes.     

The use of repetitive DNA in cytogenetic studies of plant sex chromosomes

The structure of sex chromosomes in plants was analyzed by fluorescent in situ hybridization (FISH) with repetitive DNAs. FISH probes were successfully obtained from DNA libraries that were amplified from microdissected sex chromosomes. Some probes hybridized to the subtelomeric regions, where many kinds of repetitive DNAs are located with intrachromosomal similarity of their repeat units rather than interchromosomal similarity. For example, FISH with the subtelomeric repetitive sequence can easily show the location of the pseudoautosomal region (PAR) on the X chromosome of Silene latifolia. The other probes were localized on the interstitial region of the sex chromosomes. The interstitial region contains chloroplast DNAs or neighboring sequences of the internal telomeres, suggesting insertion or translocation occurred during differentiation of the sex chromosome. These data are very informative for understanding the structure of the plant sex chromosomes and their evolutionary process.     

A video image correlation technique for the measurement of electrophoretic mobility

The electrophoretic movement of blood cells has been examined with a video image correlator using the signal from a 625 line monochrome television camera attached to the optical arrangement. The diffraction pattern of a cell produces a characteristic signal which can be detected and registered in any television scan line to form the data of the first image frame. Under the influence of an electric field, cells moved across the television field of view and after a short interval appeared on different scan lines. The detection and registering process was repeated, and a cross-correlation function between the first and second image frame was computed. This function was stored in an output buffer which was continuously updated as fresh functions were integrated with the existing contents. The mean electrophoretic mobility of a suspension of particles can be calculated from the peak position of the integrated correlation function. Using a triangle deconvolution procedure, the relative contributions of subpopulations lymphocytes prepared from peripheral blood samples were examined.     

The effect of immune complexes on the electrophoretic mobility of sheep erythrocytes

Influence of concentration of aggregated immunoglobulins with varying molecular weights on the electrophoretic mobility of ram erythrocytes has been explored. It was shown, that electrophoretic mobility of ram erythrocytes depend on the quantity and the size of adsorption complexes.     

The influence of unipolar air ions on the electrophoretic mobility of erythrocytes

Positive air ions decreased the electrophoretic mobility of erythrocytes of cats while negative air ions increased it. Inhalation of normal air supplied by a fan did not affect the electrophoretic mobility of erythrocytes.

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Zusammenfassung Positive Luftionen setzten die elektrophoretische Beweglichkeit der Erythrocyten von Katzen herab, während negative Luftionen sie steigerten. Die Inhalation normaler Luft, die von einem Ventilator unterstüzt wurde,beeinflusste die elektrophoretische Beweglichkeit von Erythrocyten nicht.

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Resume Les ions positifs diminuent la mobilité électrophorétique des érythrocytes des chats, alors que les ions négatifs l'augmentent. L'inhalation d'air normal,même activée par un ventilateur, ne l'influence par contre pas.

     

The electrophoretic mobility of gram-negative and gram-positive bacteria: an electrokinetic analysis.

The electrophoretic mobility (EPM) of a variety of Gram-negative and Gram-positive bacteria was measured with a Penkem S3000 analyser. Under standard growth conditions and neutral pH all cells displayed a negative EPM. The polysaccharide capsules of Escherichia coli strains K1, K5, K29 and K30 generated the highest EPM; to a lesser and varying degree O-antigens with charged groups and core lipopolysaccharides also contribute to the net EPM. Very little negative EPM was measured in suspension cultures of the gliding bacterium Cytophaga U67. No difference in the EPM was observed between rapidly growing and stationary-phase E. coli B. De-energization of the cell membranes by carbonyl cyanide m-chlorophenylhydrazone (CCCP) did not affect the EPM of wild-type and deep rough mutants of E. coli; and the EPM of Cytophaga U67 and Acholeplasma laidlawii remained unaltered by CCCP when measured in their respective growth media. Extrusion of filamentous bacteriophage f1 from cells of its host, E. coli A95, caused a shift to a higher negative EPM. We also measured a variety of Gram-positive strains, all of which displayed different EPMs. When membrane fractions of E. coli were adsorbed to latex spheres, characteristic differences between the EPM of beads coated with either inner or outer membrane were observed. The results suggest that the rapid EPM analysis is a useful tool to study the net electric charge of microorganisms and to examine changes of surface properties during interaction of cells with viruses, proteins (antibody) and charged antibiotics.