Tuneko Okazaki

Construction of YAC-based mammalian artificial chromosomes

To construct a mammalian artificial chromosome (MAC), telomere repeats and selectable markers were introduced into a 100 kb yeast artificial chromosome (YAC) containing human centromeric DNA. This YAC, which has a regular repeat structure of alpha-satellite DNA and centromere protein B (CENP-B) boxes, efficiently formed MACs that segregated accurately and bound CENP-B, CENP-C, and CENP-E. The MACs appear to be about 1–5 Mb in size and contain YAC multimers. Structural analyses suggest that the MACs have not acquired host sequences and were formed by a de novo mechanism. The accurate segregation of the MACs suggests they have potential as vectors for introducing genes into mammals.

CENP-A, -B, and -C Chromatin Complex That Contains the I-Type α-Satellite Array Constitutes the Prekinetochore in HeLa Cells

ABSTRACT CENP-A is a component of centromeric chromatin and defines active centromere regions by forming centromere-specific nucleosomes. We have isolated centromeric chromatin containing the CENP-A nucleosome, CENP-B, and CENP-C from HeLa cells using anti-CENP-A and/or anti-CENP-C antibodies and shown that the CENP-A/B/C complex is predominantly formed on α-satellite DNA that contains the CENP-B box (αI-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the αI-type array as a result of interactions between CENP-B and CENP-B boxes, implying a repetitive configuration for the CENP-B/CENP-A nucleosome complex. Molecular mass analysis by glycerol gradient sedimentation showed that MNase digestion released a CENP-A/B/C chromatin complex of three to four nucleosomes into the soluble fraction, suggesting that CENP-C is a component of the repetitive CENP-B/CENP-A nucleosome complex. Quantitative analysis by immunodepletion of CENP-A nucleosomes showed that most of the CENP-C and approximately half the CENP-B took part in formation of the CENP-A/B/C chromatin complex. A kinetic study of the solubilization of CENPs showed that MNase digestion first released the CENP-A/B/C chromatin complex into the soluble fraction, and later removed CENP-B and CENP-C from the complex. This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENP-C through interaction with DNA. On the basis of these results, we propose that the CENP-A/B/C chromatin complex is selectively formed on the I-type α-satellite array and constitutes the prekinetochore in HeLa cells.

Construction and characterization of the deletion mutant of hupA and hupB genes in Escherichia coli.

Insertion and deletion mutations of the hupB and hupA genes, which encode the HU-1 and HU-2 proteins, respectively, of Escherichia coli, have been constructed in vitro and transferred to the hup loci on the bacterial chromosome. The mutations were constructed by inserting a gene encoding chloramphenicol resistance or kanamycin resistance into the coding region of the hupB or hupA gene, respectively. A complete deletion of the hupA gene was constructed by replacing the entire hupA coding region with the kanamycin resistance gene. Cells in which either the hupB or the hupA gene is defective grow normally, but cells in which both of the hup genes are defective exhibit phenotypes different from the wildtype strain. The hupA-hupB double mutants are cold-sensitive, although their growth rate is normal at 37 degrees C. Furthermore, the viability of the hupA-hupB double mutants is severely reduced when the cells are subjected to either cold shock or heat shock, indicating that the hup genes are essential for cell survival under some conditions of stress. The double mutants also exhibit filamentation when grown in the lower range of permissive growth temperature.

A novel DnaA protein-binding site at 94.7 min on the Escherichia coli chromosome.

There is a DNA sequence which has unusually high affinity for the DnaA protein of Escherichia coli between the glyV and amiB–mutL operons at 94.7 min on the genetic map. Affinity of DnaA protein for DNA was measured in vivo as the activity of β‐galactosidase produced from the lacZ gene on a plasmid fused to the 5′‐terminal portion of the mioC gene, which is under the control of the DnaA protein. The chromosomal DNA segment between the two operons, carried on a compatible plasmid, derepressed the β‐galactosidase activity by titrating DnaA protein. Derepression occurred on the chromosomal dnaA gene as well, since it is autoregulated. Hence, as measured by immunoassays, one plasmid molecule carrying the DnaA‐binding region titrated 370 DnaA molecules, which is a value eightfold higher than that for a plasmid containing the oriC region. We estimate that about 60% of the total cellular DnaA molecules are bound to this site. Four DnaA‐binding sequences (DnaA boxes) and a DnaA‐regulated promoter directing transcription of two small genes were present within a 250 bp stretch in this region but additional long DNA regions, including the fifth DnaA box located about 650 bp downstream, were required for maximum binding. A role for the DnaA‐binding site in controlling DnaA‐protein concentration in the cell cycle is discussed.

Mechanism of DNA chain growth: XVI. Analyses of RNA-linked DNA pieces in Escherichia coli with polynucleotide kinase

An improved method for the isolation of RNA-linked DNA pieces from Escherichia coli has been developed. The following results, obtained by end group labelling with T4 polynucleotide kinase, strongly suggest that the short RNA segment is covalently linked to the 5′ end of DNA. (1) After denaturation by dimethyl sulphoxide, all the labelled RNA bands at the position of DNA in a Cs 2 SO 4 equilibrium density gradient. (2) The number of 5′ termini of RNA equals the number of 5′-hydroxyl termini of DNA produced from the same preparation by alkaline hydrolysis. (3) After digestion with pancreatic DNAase, the 5′-terminally labelled RNA is resistant to periodate oxidation. This suggests that these molecules contain deoxyribonucleotides at their 3′ termini. The size of the majority of the RNA segments thus obtained ranges from mono- to trinucleotides. The cellular abundance of the RNA-linked DNA pieces has been estimated by selective polynucleotide kinase-catalysed labelling of the 5′-hydroxyl ends of DNA generated by alkaline hydrolysis. In the restrictive conditions RNA-linked DNA pieces accumulate in mutants defective in the 5′ → 3′ exonuclease and/or the polymerase activity of DNA polymerase I, but not in a DNA ligase mutant or in the wild-type control. This suggests that the removal of the RNA attached to the nascent DNA pieces requires the concerted action of both the 5′ → 3′ exonuclease and the polymerase activities of DNA polymerase I. The RNA-linked DNA pieces were hydrolysed with alkali and incubated with polynucleotide kinase and [γ- 32 P]ATP. When the DNA thus labelled is degraded to 5′-mononucleotides, the 32 P is found in all four deoxyribonucleotides.

Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle

In this study, we have examined a DNA element specific to the centromere domain of human chromosomes. Purified HeLa chromosomes were digested with the restriction enzyme Sau3AI and fractionated by sedimentation through a sucrose gradient. Fractions showing antigenecity to anticentromere (kinetochore) serum obtained from a scleroderma CREST patient were used to construct a DNA library. From this library we found one clone which has specifically hybridized to the centromere domain of metaphase chromosomes using a biotinylated probe DNA and FITC-conjugated avidin. The clone contained a stretch of alphoid DNA dimer. To determine precisely the relative location of the alphoid DNA stretch and the centromere antigen, a method was developed to carry out in situ hybridization of DNA and indirect immunofluorescent staining of antigen on the same cell preparation. Using this method, we have found perfect overlapping of the alphoid DNA sites with the centromere antigen sites in both metaphase chromosomes and nuclei at various stages in the cell cycle. We have also observed this exact correlation at the attachment sites of artificially extended sister chromatids. These results suggest the possibility that alphoid DNA repeats are a key component of kinetochore structure.

Assay of centromere function using a human artificial chromosome

Abstract. In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the α21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1–5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the α21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that α21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes.

Function of RNase H in DNA replication revealed by RNase H defective mutants of Escherichia coli

SummaryEscherichia coli rnh mutants were isolated using localized mutagenesis and selective measurements of RNase H activity in mutagenized cell extracts with [3H]poly(rC)·poly(dG) as substrate. RNase H activity in extracts of one mutant, ON152 (rnh-91), was undetectable (less than 0.05% of that of wild-type cells). This mutant formed small colonies at 43 °C. At this temperature, accumulation of nascent fragments was more prominent in the rnh-91·polA4113 double mutant than in the polA4113 mutant; however, no accumulation was found in the rnh single mutant at 43° C. Unlike the 1–3 nucleotide primer RNA found on nascent fragments of polA4113 cells, primers from the rnh-91·polA4113 cells ranged from one to about ten bases. These results suggest that the 5′→3′ exonuclease activity of DNA polymerase I plays a major role in removal of primer RNA and that RNase H functions in an auxiliary role, excising the 5′-portion of longer primers.The rnh mutant supports replication of ColE1-type plasmids. A possible mechanism of replication of such plasmids in rnh mutants and a role of RNase H in the initiation of chromosomal replication are discussed.

Analysis of protein-DNA and protein-protein interactions of centromere protein B (CENP-B) and properties of the DNA-CENP-B complex in the cell cycle.

We previously reported that centromere protein B (CENP-B) forms a stable complex (designated complex A) containing two alphoid DNAs in vitro. Domains in the CENP-B polypeptide involved in the formation of complex A were determined in the present study with truncated derivatives expressed in Escherichia coli and in rabbit reticulocyte lysates. It was revealed by gel mobility shift analyses that polypeptides containing the NH2-terminal DNA-binding domain bind a DNA molecule as a monomer, while dimerizing at a novel hydrophobic domain in the COOH-terminal region of 59 amino acid residues. This polypeptide dimerization activity at the COOH-terminal region was also confirmed with the two-hybrid system in Saccharomyces cerevisiae cells. The results thus proved that CENP-B polypeptides form a homodimer at the COOH-terminal hydrophobic domain, each binding a DNA strand at their NH2-terminal domains. The dimerization and DNA-binding domains fall into two of the three completely conserved sequences found in human and mouse CENP-B, and complex A-forming activity was also detected in nuclear extracts of mouse cells. Metaphase-specific phosphorylation of CENP-B was also detected, but this had no effect on its complex A-forming activity. On the basis of the present results, we propose that CENP-B plays an important role in the assembly of specific centromere structures by forming unique DNA-protein complexes at the sites of CENP-B boxes on the centromeric repetitive DNA both in interphase nuclei and on mitotic chromosomes.

Human Artificial Chromosomes Constructed Using the Bottom-up Strategy Are Stably Maintained in Mitosis and Efficiently Transmissible to Progeny Mice

Human artificial chromosomes (HACs) are alternative vectors that promise to overcome problematic transgene expression often occurring with conventional vectors in mammalian cells and bodies. We have successfully generated HACs by multimerization of a cloned long alphoid stretch in a human cell line, HT1080. Furthermore, we developed technologies for cloning large genomic regions into HACs by means of co-transfection of clones with the alphoid array and clones encoding the genomic region of interest. The purpose of this study was to investigate the mitotic and meiotic stability of such HACs in mouse cells and bodies. We transferred a circular HAC containing the guanosine triphosphate cyclohydolase I gene (GCH1-HAC) and a linear HAC containing the human globin gene cluster (globin-HAC) from HT1080 cells into mouse embryonic stem (ES) cells by microcell-mediated chromosome transfer. The HACs were stably maintained in mouse ES cells for 3 months. GCH1-HACs in every ES cell line and globin-HACs in most ES cell lines maintained their structures without detectable rearrangement or acquisition of mouse genomic DNA except one globin-HAC in an ES cell line rearranged and acquired mouse-type centromeric sequences and long telomeres. Creation of chimeric mice using ES cells containing HAC and subsequent crossing showed that both the globin-HAC that had rearranged and acquired mouse type centromeric sequences/long telomeres and GCH1-HACs were retained in tissues of mice and transmitted to progeny. These results indicate that human artificial chromosomes constructed using the bottom-up strategy based on alphoid DNA are stable in mouse bodies and are transmissible.