Chromosomal localization of 9 KOX zinc finger genes: physical linkages suggest clustering of KOX genes on chromosomes 12, 16, and 19

Nine KOX zinc finger genes were localized on four human chromosomes by in situ hybridization of cDNA probes to metaphase chromosomes. KOX1 (ZNF10), KOX11 (ZNF18), and KOX12 (ZNF19) were mapped to chromosome bands 12q24.33, 17p13-p12, and 16q22-q23, respectively. Six other KOX genes were localized on chromosome 19: KOX6 (ZNF14) and KOX13 (ZNF20) to 19p13.3-p13.2, KOX5 (ZNF13) and KOX22 (ZNF27) to 19q13.2-qter, and KOX24 (ZNF28) and KOX28 (ZNF30) to 19q13.4. Pulsed field gel electrophoresis experiments showed that the pairs of KOX genes found on the chromosome bands 12q24.33, 16q22-q23, 19p13.3-p13.2, or 19q13.3-qter lie within 200–300 kb DNA fragments. This suggests the existence of KOX gene clusters on these chromosomal bands.     

Chromosomes of kinetoplastida

We have compared chromosome-sized DNA molecules (molecular karyotypes) of five genera (nine species) of kinetoplastida after cell lysis and deproteinization of DNA in agarose blocks and size fractionation of the intact DNA molecules by pulsed field gradient (PFG) gel electrophoresis. With the possible exception of Trypanosoma vivax and Crithidia fasciculata, all species have at least 20 chromosomes. There are large differences between species in molecular karyotype and in the chromosomal distribution of the genes for alpha- and beta-tubulin, rRNA and the common mini-exon sequence of kinetoplastid mRNAs. In all cases, the rRNA genes are in DNA that is larger than 500 kb. Whereas T. brucei has approximately 100 mini-chromosomes of 50-150 kb, only few are found in T. equiperdum; T. vivax has no DNA smaller than 2000 kb. As all three species exhibit antigenic variation, small chromosomes with telomeric variant surface glycoprotein genes cannot be vital to the mechanism of antigenic variation. The apparent plasticity of kinetoplastid genome composition makes PFG gel electrophoresis a potentially useful tool for taxonomic studies.     

Construction of a Not I restriction map of the fission yeast Schizosaccharomyces pombe genome.

Pulsed field gel electrophoresis and large DNA technology were used to construct a Not I restriction map of the entire genome of the fission yeast Schizosaccharomyces pombe. There are 14 detectable Not I sites in S. pombe 972h: 9 sites on chromosome I and 5 sites on chromosome II, while no Not I sites were found on chromosome III. The 17 fragments (including intact chromosome III) generated by Not I digestion were resolved by PFG electrophoresis. These fragments ranged in size from 4.5 kb to approximately 3.5 Mb. Various strategies were applied in determining, efficiently, the order of the fragments on the chromosomes. The genomic size measured by adding all the fragments together is about 14 Mb and the sizes of the three chromosomes are I, 5.7 Mb, II, 4.6 to 4.7 Mb, and III, 3.5 Mb. These are generally somewhat smaller than estimated previously.     

Identification and isolation of Cryptosporidium parvum genes encoding microtubule and microfilament proteins.

Microtubules and microfilaments are highly conserved cytoskeletal polymers hypothesized to play essential biomechanical roles in the unusual gliding motility of Apicomplexan zoites and in their invasion of, and development within, host epithelial cells. We have identified and isolated Cryptosporidium parvum genes encoding the microtubule proteins alpha- and beta-tubulin and the microfilament protein actin by screening a lambda gt11 C. parvum genomic DNA library with degenerate oligonucleotide and heterologous cDNA hybridization probes respectively. The alpha- and beta-tubulin genes have been partially sequenced and the deduced peptide sequences show greatest homology with the tubulins of the related parasites, T. gondii and P. falciparum. The complete nucleic acid sequence of the actin gene predicts a 376 amino acid, 42 kDa protein having 85% sequence identity with the P. falciparum actin I and the human gamma-actin proteins. Each of these cytoskeletal protein genes was demonstrated to be of cryptosporidial origin by Southern analyses of C. parvum chromosomes fractionated by pulsed field gel electrophoresis; the cloned alpha- and beta-tubulin genes hybridized with chromosomes of ca. 1,200 and 1,500 kb respectively and the cloned actin gene also hybridized with a 1,200 kb chromosome.     

Electrophoretic karyotype and linkage groups of the amoeboflagellate Naegleria gruberi

We have constructed a molecular karyotype for two strains of Naegleria gruberi using pulsed field gel electrophoresis. Each strain has about 23 chromosomes, considerably more than any previous estimate. These chromosomes range in size from 400 kilobasepairs to over 2,000 kilobasepairs. In Naegleria, construction of the DNA karyotype depends on assessment of the anomalous electrophoretic mobility of the circular ribosomal RNA genes. We have determined the chromosomal locations of an identified unique gene (flagellar calmodulin) and four identified multigene families (alpha- and beta-tubulin, actin, ubiquitin), as well as three differentially expressed genes of unknown functions. The ca. 12 actin genes are dispersed over at least seven chromosomes, whereas the majority of the more than eight alpha-tubulin genes are confined to a single chromosome. The ubiquitin genes are found on five chromosomes in one strain and seven in the other and the beta-tubulin genes are on three or four. Our observations provide a foundation for molecular genetic studies in this organism.     

Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.     

Structure and arrangement of the beta-tubulin genes of Leishmania tropica.

We studied the organization and arrangement of the genes encoding beta-tubulin in the protozoan parasite Leishmania tropica and examined the structure and orientation of the beta-tubulin mRNA relative to the gene. There were found to be eight to nine beta-tubulin genes arranged in an array of direct tandem repeat units with a length of 3.8 kilobase pairs, and they were extremely homologous, if not identical, in sequence. These repeat units did not contain the alpha-tubulin genes. The transcribed sequences within the beta-tubulin genes were localized, and the orientation of the major alpha-tubulin mRNA was mapped on the gene by S1 nuclease analysis.     

Construction of NotI restriction map of the Streptococcus mutans genome

Streptococcus mutans and Streptococcus sobrinus are the major causative organisms of human dental caries. Pulsed-field gel electrophoresis (PFG) showed that the restriction enzyme NotI produced ten and six DNA fragments from the genomes of S. mutans strain MT8148 and S. sobrinus strain 6715, respectively. The sizes of the chromosomes of S. mutans and S. sobrinus were each estimated to be about 2200 kb. The NotI restriction map of S. mutans MT8148 genome was constructed by Southern blot analysis with probes that overlapped two adjacent NotI fragments. Several virulence-associated genes of S. mutans were placed on the NotI restriction map. In addition, unique 'fingerprints' of S. mutans chromosomal DNA digested with NotI were produced by PFG, and these may be useful for epidemiological studies.     

Mapping of human H chain V region genes (VH4) using deletional analysis and pulsed field gel electrophoresis.

We have used molecular genetic mapping techniques to establish the order and approximate chromosomal locations of VH4 elements on both chromosomes 14 from a single patient. A total of 10 BglII restriction fragments carrying VH4 elements was characterized. The genomic order of VH4-carrying restriction fragments was determined by analysis of the pattern of loss of hybridizing bands from the genomes of a panel of monoclonal lymphoblastoid cell lines which had well characterized rearrangements of the Ig locus on each chromosome. Some individual elements were identified using sequence-specific oligonucleotide probes. Physical dimensions were estimated by the assignment of these ordered elements to large (50-350-kb) restriction fragments using two-dimensional pulse field gel electrophoresis. Six such fragments spanning approximately 890 kb were physically linked and ordered. The chromosomes differed with respect to the complement of VH4 elements, although no evidence was found of major differences in organization. The establishment of a panel of well characterized deletion lines facilitates the rapid mapping of defined restriction fragments carrying VH elements.     

Chromosome Polymorphism of the Obligate Biotrophic Parasite Plasmodiophora brassicae

Electrophoretic karyotypes of different isolates of Plasmodiophora brassicae, the causative agent of clubroot of cabbage, using contour‐clamped homogeneous electric field gel electrophoresis (CHEF) revealed chromosome polymorphism in this obligate parasite. Ribosomal RNA (rRNA) genes have been localized on three chromosomes (I, IV and V) of P. brassicae of the 16 chromosomal bands, which can be distinguished for the single‐spore isolate ‘e₃’. In comparison with this isolate three other single‐spore isolates showed chromosome polymorphism by size of chromosomal bands and by hybridization pattern with rRNA gene fragments and other Plasmodiophora‐specific DNA fragments.     

Resolution of Acanthamoeba castellanii chromosomes by pulsed field gel electrophoresis and construction of the initial linkage map

Pulsed field gel electrophoresis has been used to resolve chromosome-sized DNA molecules in fungi and parasites but has not yet been used successfully to examine the chromosomes of other lower eukaryotes used extensively for biochemical research such as Acanthamoeba, Physarum, and Dictyostelium. Here we show an electrophoretic karyotype of the protozoan Acanthamoeba castellanii using orthogonal field alternating gel electrophoresis (OFAGE). There are about 20 small chromosomes ranging in size from 220 kb to >2 Mb. We have assembled initial linkage groups assigning all of the cloned Acanthamoeba genes to chromosome-sized DNA molecules. Actin, suggested to have three or more non-allelic genes, maps to at least eight distinct chromosome bands. Two myosin II genes localize to two different chromosomal bands while myosin IB and 18S rRNA map to unresolved larger chromosomes.Abbreviations OFAGE Orthogonal field alternating gel electrophoresis     

Genomic organization of the halophilic archaeon Haloferax mediterranei: physical map of the chromosome.

Pulsed field gel electrophoresis (PFG) has been used to study the genomic organization of the halophilic archaeon Haloferax mediterranei. Analysis of the different genomic elements as well as the restriction patterns obtained with several endonucleases revealed that this microorganism has a circular chromosome of 2.9 Mb and, at least, three extrachromosomal elements of 490, 320 and 130 kb respectively. The complete physical map of the chromosome for the endonucleases PacI and BamHI has been constructed, and several BcII, BgIII and DraI restriction fragments have been aligned on these maps. The localization of heterologous and homologous genes on the physical map, including those for rRNA, lay the ground work for the construction of a genetic map.     

Molecular karyotype analysis and mapping of housekeeping genes to chromosomes of selected species complexes of Leishmania.

The molecular karyotypes for 20 reference strains of species complexes of Leishmania were determined by contour-clamped homogeneous electric field (CHEF) electrophoresis. Determination of number/position of chromosome-sized bands and chromosomal DNA locations of housekeeping genes were the two criteria used for differentiating and classifying the Leishmania species. We have established two gel running conditions for optimal separation of chromosomes, which resolved DNA molecules as large as 2,500 kilobase pairs (kb). Chromosomes were polymorphic in number (22-30) and size (200-2,500 kb) of bands among members of five complexes of Leishmania. Although each stock had a distinct karyotype, in general the differences found between strains and/or species within each complex were not clear enough for parasite identification. However, each group showed a specific number of size-concordant DNA molecules, which allowed distinction among the Leishmania complex parasites. Clear differences between the Old and New world groups of parasites or among some New World Leishmania species were also apparent in relation to the chromosome locations of beta-tubulin genes. Based on these results as well as data from other published studies the potential of using DNA karyotype for identifying and classifying leishmanial field isolates is discussed.     

Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes.

A macrorestriction map representing the complete physical map of the Rhodobacter sphaeroides 2.4.1 chromosomes has been constructed by ordering the chromosomal DNA fragments from total genomic DNA digested with the restriction endonucleases AseI, SpeI, DraI, and SnaBI. Junction fragments and multiple restriction endonuclease digestions of the chromosomal DNAs derived from wild-type and various mutant strains, in conjunction with Southern hybridization analysis, have been used to order all of the chromosomal DNA fragments. Our results indicate that R. sphaeroides 2.4.1 carries two different circular chromosomes of 3,046 +/- 95 and 914 +/- 17 kilobases (kb). Both chromosome I (3,046 kb) and chromosome II (914 kb) contain rRNA cistrons. It appears that only a single copy of the rRNA genes is contained on chromosome I (rrnA) and that two copies are present on chromosome II (rrnB, rrnC). Additionally, genes for glyceraldehyde 3-phosphate dehydrogenase (gapB) and delta-aminolevulinic acid synthase (hemT) are found on chromosome II. In each instance, there appears to be a second copy of each of these genes on chromosome I, but the extent of the DNA homology is very low. Genes giving rise to enzymes involved in CO2 fixation and linked to the gene encoding the form I enzyme (i.e., the form I region) are on chromosome I, whereas those genes representing the form II region are on chromosome II. The complete physical and partial genetic maps for each chromosome are presented.     

Localization of the active gene of aldolase on chromosome 16, and two aldolase A pseudogenes on chromosomes 3 and 10

Summary Southern blot analysis of human genomic DNA hybridized with a coding region aldolase A cDNA probe (600 bases) revealed four restriction fragments with EcoRI restriction enzyme: 7.8 kb, 13 kb, 17 kb and >30 kb. By human-hamster hybrid analysis (Southern technique) the principal fragments, 7.8 kb, 13 kb, >30 kb, were localized to chromosomes 10, 16 and 3 respectively. The 17-kb fragment was very weak in intensity; it co-segregated with the >30-kb fragment and is probably localized on chromosome 3 with the >30-kb fragment. Analysis of a second aldolase A labelled probe protected against S1 nuclease digestion by RNAs from different hybrid cells, indicated the presence of aldolase A mRNAs in hybrid cells containing only chromosome 16. Under the stringency conditions used, the EcoRI sequences detected by the coding region aldolase A cDNA probe did not correspond to aldolase B or C. The 7.8-kb and >30-kb EcoRI sequences, localized respectively on chromosomes 10 and 3, correspond to aldolase A pseudogenes, the 13-kb EcoRI sequence localized on chromosome 16 corresponds to the aldolase active gene. The fact that the aldolase A gene and pseudogenes are located on three different chromosomes supports the hypothesis that the pseudogenes originated from aldolase A mRNAs, copied into DNA and integrated in unrelated chromosomal loci.     

Pig sperm tail tubulin. Its extraction and characterization

Axonemal tubulin extracted from pig sperm tails has been characterized by one- and two-dimensional electrophoresis and by one-dimensional peptide mapping. The electrophoretic mobilities of its subunits after reduction and carboxymethylation were similar to those of the major subunits of pig brain tubulin. Sperm tail tubulin subunits also had roughly the same isoelectric points as pig brain tubulin subunits, except that they appeared to have a relatively larger tailing effect. The proteolytic cleavage pattern of the pig sperm tail beta-tubulin closely resembled those of both the tunicate (Ciona intestinalis) sperm beta-tubulin and pig brain beta-tubulin. The peptide pattern of pig sperm tail alpha-tubulin, however, was more similar to that of tunicate sperm tail alpha-tubulin than to that of pig brain alpha-tubulin. This supports the hypothesis put forward in a previous investigation [1] that functionally similar tubulins from taxonomically distant species can be more related than functionally dissimilar tubulins from the same species.