Ors12, a mammalian autonomously replicating DNA sequence, is present at the centromere of CV-1 cell chromosomes

ors12, an 812-bp-long sequence, previously isolated by extrusion of nascent DNA from replication bubbles active at the onset of S phase (G. Kaufmann, M. Zannis-Hadzopoulus, and R. G. Martin Mol. Cell. Biol. 5, 721-727, 1985), has been shown to function as an origin of DNA replication in autonomously replicating plasmids (L. Frappier and M. Zannis-Hadjopoulos Proc. Natl. Acad. Sci. USA 84, 6668-6672, 1987) and in a cell-free system (C. E. Pearson, L. Frappier, and M. Zannis-Hadzopoulos Biochim. Biophys. Acta 1090, 156-166, 1991). A portion of ors12 (nucleotides 1-168) consists of the highly reiterated alpha-satellite sequence (B. S. Rao et al. Gene 87, 233-242, 1990). We have estimated the copy number of the non-alpha-satellite portion of ors12 in CV-1 cells to be < 9 copies per haploid genome and have used it as a probe to generate a genomic map of ors12 on CV-1 DNA. In situ hybridization of CV-1 metaphase chromosomes, using a biotinylated probe of the entire ors12 sequence, positively identified the centromeres of all chromosomes. However, when the non-alpha-satellite portion of ors12 was used as a probe, it positively identified the centromeric region of only six chromosomes, namely, B4, C11, D14, D24, E25, and E27, as well as that of a marker chromosome. The results suggest that ors12 represents a centromeric putative replication origin that is present on a subset of CV-1 chromosomes and is activated at the onset of S phase.     

Cloning, expression, and nucleotide sequence of the Escherichia coli K-12 ackA gene.

The Escherichia coli K-12 ackA gene, which encodes an acetate kinase, was cloned. The acetate kinase activities of ackA+ plasmid-containing strains were amplified 160- to 180-fold. The complete nucleotide sequence of the ackA gene was determined. It was deduced that the ackA gene coded for a protein of 400 amino acids with an Mr of 43,297. The ackA gene was found to be located about 15 kilobases upstream of the purF-folC-hisT region of the chromosome.     

Comparison of the maxicircle (mitochondrial) genomes of Leishmania tarentolae and Trypanosoma brucei at the level of nucleotide sequence

The entire 16.7-kilobase (kb) transcribed region of the Leishmania tarentolae maxicircle was compared to the entire 15-kb transcribed region of the Trypanosoma brucei maxicircle at the nucleotide sequence level by dot matrix analysis and by alignments of individual genes. The L. tarentolae NADH dehydrogenase subunit 1 (ND1) gene was identified in a newly obtained 2.9-kb sequence. All but two regions which flank the cytochrome b gene are highly conserved in both species. One 3.1-kb region in L. tarentolae that contains the cytochrome oxidase subunit III (COIII) gene and several open reading frames corresponds to a 2-kb sequence in T. brucei with limited sequence homology that lacks the COIII gene. Another 0.6-kb region that comprises an unidentified open reading frame (open reading frame 12) in L. tarentolae is substituted by a nonhomologous 0.4-kb open reading frame in T. brucei. A short intergenic region between the ND1 gene and the maxicircle unidentified reading frame 1 gene shows limited sequence homology, and the regions between the ND4 and ND5 genes and between the COI and ND4 genes are not conserved. All of the intergenic regions share G + C richness and a similar pattern of G versus C strand bias. 1.8 kb of the L. tarentolae divergent region (DV) and around 3 kb of the T. brucei DV were also obtained. The T. brucei DV sequences were not homologous to the L. tarentolae DV sequence but were organized in a similar fashion with tandem repeats of varying complexity.     

Cloning and complete sequence of an HLA-A2 gene: analysis of two HLA-A alleles at the nucleotide level

The gene for the HLA-A2 antigen has been cloned from the human lymphoblastoid cell line 721. Comparison of this sequence with the published sequence for HLA-A3 permits the examination of two alleles at the extremely polymorphic HLA-A locus. A high degree of sequence conservation was seen in both coding and noncoding DNA, 97.2% and 94.5%, respectively. Interestingly, the 3' untranslated region was the most conserved, with 99.5% homology. The polymorphism of the HLA-A antigens results from a high proportion of amino acid substitutions relative to the total nucleotide changes in exons 2 and 3. Unlike the clustered differences seen in this region on comparison of two H-2K alleles of mouse, nucleotide substitutions between the HLA-A2 and A3 alleles are evenly distributed. Substitutions at silent sites and within introns were used to calculate an intra-allelic divergence time of at least 10 to 15 million years for these two HLA-A alleles.     

Nucleotide sequence comparison of a chromosome rearrangement on human chromosome 12 and the corresponding ape chromosomes

Chromosome rearrangement has been considered to be important in the evolutionary process. Here, we demonstrate the evolutionary relationship of the rearranged human chromosome 12 and the corresponding chromosome XII in apes (chimpanzee, bonobo, gorilla, orangutan, and gibbon) by examining PCR products derived from the breakpoints of inversions and by conducting shotgun sequencing of a gorilla fosmid clone containing the breakpoint and a "duplicated segment" (duplicon). We confirmed that a pair of 23-kb duplicons flank the breakpoints of inversions on the long and short arms of chimpanzee chromosome XII. Although only the 23-kb duplicon on the long arm of chimpanzee chromosome XII and its telomeric flanking sequence are found to be conserved among the hominoids (human, great apes, and gibbons), the duplicon on the short arm of chimpanzee chromosome XII is suggested to be the result of a duplication from that on the long arm. Furthermore, the shotgun sequencing of a gorilla fosmid indicated that the breakpoint on the long arm of the gorilla is located at a different position 1.9 kb from that of chimpanzee. The region is flanked by a sequence homologous to that of human chromosome 6q22. Our findings and sequence analysis suggest a close relationship between segmental duplication and chromosome rearrangement (or breakpoint of inversion) in Hominoidea. The role of the chromosome rearrangement in speciation is also discussed based on our new results.     

The size and form of chromosomes are constant in the nucleus, but highly variable in bacteria, mitochondria and chloroplasts

From cytological examination, the size and form of the chromosomes in the eukaryotic nucleus are invariant across generations, leading to the expectation that constancy of inheritance likely depends on constancy of the chromosomal DNA molecule conveying the constant phenotype. Indeed, except for rare mutations, major phenotypic traits appear largely without change from generation to generation. Thus, when it was discovered that the inheritance of traits for bacteria, mitochondria and chloroplasts was also constant, it was assumed that chromosomes in those locations were also constant. Such has not turned out to be the case, however; those chromosomes are highly variable in structure. I propose, therefore, that only for the nucleus is there a requirement that a chromosome be "finished" (contain only fully replicated genomes) before it may segregate to daughter cells. This requirement does not apply to the variable chromosomes among chloroplasts, mitochondria and bacteria.     

Identification and characterization of repetitive DNA in the genus Didelphis Linnaeus, 1758 (Didelphimorphia,Didelphidae) and the use of satellite DNAs as phylogenetic markers

Didelphis species have been shown to exhibit very conservative karyotypes, which mainly differ in their constitutive heterochromatin, known to be mostly composed by repetitive DNAs. In this study, we used genome skimming data combined with computational pipelines to identify the most abundant repetitive DNA families of Lutreolina crassicaudata and all six Didelphis species. We found that transposable elements (TEs), particularly LINE-1, endogenous retroviruses, and SINEs, are the most abundant mobile elements in the studied species. Despite overall similar TE proportions, we report that species of the D. albiventris group consistently present a less diverse TE composition and smaller proportions of LINEs and LTRs in their genomes than other studied species. We also identified four new putative satDNAs (sat206, sat907, sat1430 and sat2324) in the genomes of Didelphis species, which show differences in abundance and nucleotide composition. Phylogenies based on satDNA sequences showed well supported relationships at the species (sat1430) and groups of species (sat206) level, recovering topologies congruent with previous studies. Our study is one of the first attempts to present a characterization of the most abundant families of repetitive DNAs of Lutreolina and Didelphis species providing insights into the repetitive DNA composition in the genome landscape of American marsupials.     

The genes for CD37, CD53, and R2, all members of a novel gene family,are located on different chromosomes

CD37, CD53, and R2 leukocyte surface antigens are members of a novel family of structurally related proteins. They all have four transmembrane-spanning domains with a single major extracellular loop. The CD37 is expressed on B cells and on a sub-population of T cells. The CD53 is known as a panleukocyte marker. The R2 protein is an activation antigen of T cells. The CD37, CD53, and R2 genes were assigned with the help of human/rodent somatic cell hybrids and human-specific probes to human chromosomes 19, 1, and 11, respectively. For the regional assignment, various deletion hybrids were used to map CD37 to 19p13-q13.4, CD53 to 1p12-p31, and R2 to 11p12.     

The levels of lactase and of sucrase-isomaltase along the rabbit small intestine are regulated both at the mRNA level and post-translationally.

We determined along the small intestine of young and adult rabbits the activities of lactase (LPH) and sucrase (SI), the levels of their cognate mRNAs, and examined the in vitro biosynthesis of LPH and pro-SI. Lactase activity is low in the proximal 1/3 of the intestine, whereas the mRNA levels are high. However, the rates of biosynthesis of the LPH forms correlated well with the steady-state levels of LPH mRNA in all segments, indicating that factor(s) acting post-translationally produce a decline in brush border LPH in the proximal small intestine. These factor(s) are not involved in the processing of pro-LPH to mature LPH, since the relative amounts of the various forms of LPH are almost the same along the small intestine. Unexpectedly, we find that also for SI the ratio of activity to mRNA is low in proximal intestine. The biosynthesis of pro-SI correlates with the steady-state levels of its mRNA. Hence, the steady-state levels of LPH and SI along the small intestine are regulated both by mRNA levels and by posttranslational factor(s).     

Cloning, nucleotide sequence, and characterization of mtr, the structural gene for a tryptophan-specific permease of Escherichia coli K-12.

The mtr gene of Escherichia coli K-12 encodes an L-tryptophan-specific permease. This gene was originally identified through the isolation of mutations in the 69-min region of the chromosome, closely linked to argG. Cells with lesions in mtr display a phenotype of 5-methyltryptophan resistance. The mtr gene was cloned by using the mini-Mu system. The amino acid sequence of Mtr (414 codons), deduced by DNA sequence analysis, was found to be 33% identical to that of another single-component transport protein, the tyrosine-specific permease, TyrP. The hydropathy plots of the two permeases were similar. Possible operator sites for the tyrosine and tryptophan repressors are situated within the region of DNA that is likely to be the mtr promoter.     

Heritable fragile sites on human chromosomes. XI. Factors affecting expression of fragile sites at 10q25, 16q22, and 17p12.

The fragile sites at 10q25, 16q22, and 17p12 can all be induced in lymphocyte culture by BrdU or BrdC added 6-12 hrs prior to harvest. Without induction, fra(10)(q25) is rarely expressed spontaneously, whereas fra(16)(q22) is frequently expressed spontaneously. Fra(17)(p12) is frequently expressed spontaneously but is probably expressed only after induction in some individuals. Distamycin A, netropsin, and Hoechst 33258 induced high levels of expression of fra(16)(q22) and fra(17)(p12) but did not enhance expression of fra(10)(q25). The mechanisms of induction of fra(16)(q22) by BrdU and distamycin A appear to be different, since the time of induction by BrdU reaches a maximum about 12 hrs prior to harvest whereas induction by distamycin A requires much longer exposure. The fragile sites at 10q25 and 16q22 were both induced in fibroblast culture by BrdU. Fra(17)(p12) is accepted as a fragile site because preliminary studies show that it behaves similarly in lymphocyte culture to fra(16)(q22); however, there is only limited evidence for fragility at 17p12.