Intact Chromosomes Research Articles

A Distal Heterochromatic Block Displays Centromeric Activity When Detached from a Natural Centromere

We repeatedly released a distal block of heterochromatin lacking a natural centromere in mitotic cells and assayed its segregation. At anaphase, control acentric fragments typically remained unoriented between daughter nuclei and were subsequently lost. Fragments containing the brown Dominant ( bw D ) heterochromatic element displayed regular anaphase movement upon release. These fragments were found to segregate and function based on both cytological and phenotypic criteria. We also found that intact bw D -containing chromosomes normally display occasional dicentric behavior, suggesting that bw D has centromeric activity on the intact chromosome as well. Our findings suggest that centromere competence is innate to satellite-containing blocks of heterochromatin, challenging models for centromere identity in which competence is an acquired characteristic.

CYTOKINESIS IN CELLS UNDERGOING MITOSIS WITHOUT GENOME REPLICATION

Chinese hamster ovary cells can be forced to enter mitosis without prior DNA replication by treatment with hydroxyurea and caffeine. Cells treated in this way assemble a spindle that functions normally except that it does not accomplish anaphase spindle elongation (anaphase B). The chromatin detaches from the unreplicated kinetochores, which fragment, but establish microtubule attachments and migrate to the metaphase plate. Partitioning of the kinetochore fragments ensues on the normal schedule. Typical midbodies and cleavage furrows are established and daughter cells of equal size are produced. These results imply that intact chromosomes are not necessary for correct cleavage furrow placement but that kinetochores might be. Further, it is clear that cleavage furrow placement does not depend on anaphase spindle elongation.

Identification and characterisation of three antigenic proteins from Cryptosporidium parvum sporozoites using a DNA library expressing poly-histidine tagged peptides

To identify antigenic peptides of the parasite Cryptosporidium parvum, an expression library that allows for the production of chimeric proteins fused with a 6-histidine tag was made. The library was screened with C. parvum sporozoite rabbit anti-serum, and three positive clones (sa20, sa35, and sa40) were identified. The corresponding recombinant proteins ( SA20, SA35, and SA40) were expressed in Escherichia coli and purified by metal-affinity chromatography. The sequence of sa20 and sa35 clones did not show any homology with known genes or proteins, whereas the 5′ end of the sa40 clone showed homology with two previously identified C. parvum sequences. Hybridisations to intact chromosomes fractionated by pulsed-field gel electrophoresis revealed that the sa35 and sa40 sequences are localised on chromosome VII, whereas the sa20 sequence is localised on chromosome VI. Reverse transcriptase–PCR experiments showed the presence of mRNAs for sa35 and sa40 in the oocyst, whereas the sa20 mRNA was undetectable in this stage. The serological response to the three proteins was assayed in C. parvum-immunised rabbits and in immunocompetent individuals with cryptosporidiosis. The Western blot results indicated that rabbits, challenged with a sporozoite crude antigen or with an oocyst crude antigen, were highly responsive to these three antigens. Human serum samples showed a response to the three proteins, although the response to SA20 appeared to be unrelated to a recent C. parvum infection. These results suggest that the SA35 and the SA40 proteins may be useful in detecting C. parvum infections.

The new biology enters the generalist pediatrician's office: lessons from the Human Genome Project.

1. Edward R.B. McCabe, MD, PhD* 1. 2. *Physician-in-Chief, Mattel Children’s Hospital at UCLA; Professor and Executive Chair, Department of Pediatrics, UCLA School of Medicine, Los Angeles, CA. Birth defects are the leading cause of infant mortality in the United States, representing more than 20% of all infant deaths. This infant mortality rate from birth defects exceeds that from sudden infant death syndrome, low-birthweight/short gestation, respiratory distress syndrome, and maternal complications. In addition, birth defects and genetic diseases represent major sources of morbidity for those who survive. As our ability increases to care effectively for those who have infectious diseases and other acute illnesses, individuals who have chronic illnesses due to genetic etiologies represent an increasing proportion of patients seen in the general pediatrician”s office. The Human Genome Project was initiated on October 1, 1990, and has a projected funding period of 15 years. The goal is to sequence the entire human genome, representing three billion base pairs that contain the coding sequences for approximately 75,000 genes. During the latter half of this century, investigations into the genetics of disease gathered increasing momentum. In addition to fundamental investigations into human genetics, technologic tools were developed that permitted large-scale genomic sequencing. These tools included the polymerase chain reaction (PCR), which permits amplification of hundreds of thousands or even millions of copies of DNA and requires only limited sequence data for its success; automated DNA sequencing, which allows increased sequence processing and decreased cost compared with manual methods; and improved information systems, which permit sophisticated analysis and assembly of the three billion base pairs of DNA in the human genome. Thus, the Human Genome Project represents the current chapter in our understanding, but it is neither the first nor the final chapter in this story. Once we know the sequences of all of the human genes, we must learn their functional roles in human development and disease pathogenesis. The Human Genome Project has been referred to as the “moon shot …

A transgene insertion creating a heritable chromosome deletion mouse model of Prader-Willi and angelman syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the loss of function of imprinted genes in human chromosome 15q11-q13. The central part of mouse chromosome 7 is homologous to human 15q11-q13, with conservation of both gene order and imprinted features. We report here the characterization of a transgene insertion (Epstein-Barr virus Latent Membrane Protein 2A, LMP2A) into mouse chromosome 7C, which has resulted in mouse models for PWS and AS dependent on the sex of the transmitting parent. Epigenotype (allelic expression and DNA methylation) and fluorescence in situ hybridization analyses indicate that the transgene-induced mutation has generated a complete deletion of the PWS/AS-homologous region but has not deleted flanking loci. Because the intact chromosome 7, opposite the deleted homolog, maintains the correct imprint in somatic cells of PWS and AS mice and establishes the correct imprint in male and female germ cells of AS mice, homologous association and replication asynchrony are not part of the imprinting mechanism. This heritable-deletion mouse model will be particularly useful for the identification of the etiological genes and mechanisms, phenotypic basis, and investigation of therapeutic approaches for PWS.

Resistance to Leptosphaeria maculans is conserved in a specific region of the Brassica B genome

Offspring from asymmetric hybrids between Brassica napus and the three B-genome species Brassica nigra, Brassica juncea and Brassica carinata were analysed for the presence of B-genome markers and resistance to the fungus Leptosphaeria maculans, the causal agent of blackleg disease. Twenty five plants from each species combination were analysed in the first backcross (BC1) generation, 30 plants in BC2 and 60 plants in BC3. The plants were analysed by 46 RFLP markers detecting 85 loci dispersed throughout the B. nigra genome. The plants with additional B. carinata DNA had a decrease in the presence of RFLP markers ranging from 59% in BC1 to 36% in BC2 and down to 11% in BC3. Similar results were obtained in the lines with additional DNA from B. juncea where the 60% presence of RFLP markers in BC1 was reduced to 33% in BC2 and to 10% in BC3. However presence of the markers were significantly lower in the B. nigra-derived material where BC1 had 46%, BC2 25% and BC3 8%. Since at least two loci could be detected on each end of the eight linkage groups of the B genome, the degree of symmetry was estimated. After one back-cross between 0.5 and 1.25% intact chromosomes were retained, whereas in BC2 this frequency was 0.21% for all three B-genome donor species. The maintenance of half-chromosomes ranged from 2.63% to 5.38% in BC1 and between 0.73% and 1.15% in BC2. No chromosome arms were found in any of the BC3 plants. In total, four co-segregating markers for cotyledon and adult-leaf resistance to L. maculans were found which detected six loci located on linkage groups 2, 5 and 8. When the results from the three donor species were compared, one triplicate region in the B genome had preserved the resistance loci in all three species.

Development and identification of a complete set of Triticum aestivum - Aegilops geniculata chromosome addition lines

The production and identification of a complete set of intact Aegilops geniculata chromosome and telosome additions to common wheat is described. All Ug and Mg genome chromosomes were tentatively assigned to their homoeologous groups based on C-banding, meiotic metaphase I pairing analyses and plant morphologies. Thirteen disomic and one monosomic wheat-Ae. geniculata chromosome additions were identified. Furthermore, two monotelosomic (MtA7UgL, MtA7MgL) and nine ditelosomic (DtA1UgS, DtA1UgL, DtA2UgS, DtA1MgL, DtA2MgL, DtA3MgS, DtA5MgS, DtA6MgL, DtA7MgS) wheat-Ae. geniculata additions were recovered. C-banding and meiotic pairing analyses revealed that all added Ug and Mg genome chromosomes are structurally unaltered compared to the Ae. geniculata parent accession. Chromosome 4Mg has a strong gametocidal gene that, when transferred to wheat, causes extensive chromosome breakage mainly in gametes lacking it. The relationships of Ae. geniculata chromosomes with those of the diploid progenitor species and derived polyploids is discussed.Key words: Triticum aestivum, Aegilops geniculata, chromosome addition lines, C-banding, genome evolution.

An improved method for routine preparation of intact artificial chromosome DNA (340-1000 kb) for transfection into human cells.

The transfer of high molecular weight (HMW) DNA into mammalian cells is an important strategy for assessing human gene expression and chromosome structure and function. However, using current methods, it is difficult to dependably prepare intact HMW DNA because of the susceptibility of the DNA to degradation and physical shearing. Here we describe a strategy whereby intact artificial chromosome DNA (as large as 1 Mb) can be routinely prepared from yeast. Strict adherence to this protocol has resulted in: (i) >90% of liquid DNA preparations containing largely intact DNA; (ii) transfection efficiencies for the development of stable human clonal cell lines ranging from 5 x 10(-7) to 8.8 x 10(-5); and (iii) the presence of markers from both YAC arms in 30-42% of the human fibrosarcoma cell HT1080 clones and 100% of the CF lung epithelial cell lines IB3-1 and CFT1 clones, suggesting that the HMW DNA is potentially intact in a substantial proportion of clones. Using this protocol for DNA preparation, successful transfection of functional 1 Mb human artificial chromosome DNA into human cells has also been achieved. This methodology should prove useful to those interested in using HMW human DNA for gene expression and functional analysis or for linear artificial chromosome construction, since integrity is absolutely critical for the success of these studies.

Analytical ultracentrifugation of chromatin.

The ability of analytical ultracentrifugation to elucidate chromatin structure/function relationships originates directly from its capacity to accurately measure key structural properties of complex macromolecular assemblies in solution. Figure 1 schematically illustrates the complex nature of chromatin. Newly replicated DNA is wrapped around core histone octamers spaced at approx 200 bp intervals to form nucleosomal arrays, which then interact with linker histones and numerous other nonhistone chromosomal proteins to form “chromatin.” Chromatin is conformationally dynamic, undergoing a number of short-range and long-range folding transitions to produce highly condensed interphase chromosomal fibers (Fig. 1). For short chromatin fragments studied in vitro, fiber condensation manifests both in the form of intramolecular conformational changes and reversible oligomerization (, , , ). In addition, the structure of chromatin fibers and functions such as transcription and replication are irrevocably linked; any given region of a chromosomal fiber can be either functionally active or inactive depending on both its specific complement of chromatin-associated proteins and its overall state of condensation (,). Consequently, to biochemically characterize chromatin structure/function relationships in vitro, one must be able to analyze both the intramolecular conformational dynamics and intermolecular interactions of an exceedingly complex macromolecular assembly (e.g., a 12-mer nucleosomal array containing one H1 molecule per nucleosome consists of >100 proteins and 2400 bp of DNA, has a molecular mass of approx 3.5×106 D, yet represents only roughly one millionth of an intact eukaryotic chromosome.) Open image in new window Fig. 1. Schematic illustration of the hierarchical relationships between DNA, chromatin, and interphase chromosomal fibers.

Phenotypic effect correlating with loss of a novel tumor suppressor gene: towards cloning by complementation.

We intend to use a gene complementation approach to clone a tumor suppressor gene on mouse chromosome 2, the loss of which contributes to myeloid leukemia. An in vitro model system has been generated using a clonal cell line, in which tumorigenic chromosomal lesions have been selected along with myeloid differentiation. Among these lesions are deletions of chromosome 2. Comparison of subclones with deleted vs intact chromosomes 2 has allowed the identification of a growth related phenotypic pattern which correlates with the deletion, viz the retention of a marker of immature cells, resistance to inhibition by lipopolysaccharide (LPS), even in the presence of markers of mature myeloid cells, such as resistance to killing by apoptosis-inducing agents. The phenotype is shared by chromosome 2-deleted cell lines derived from conventional tumors. We have begun to investigate the mechanism of the phenotype. The LPS resistance does not correlate with lack of mRNA for CD14, a known cell surface receptor for this agent, or with failure to induce TNF alpha or nitric oxide synthase in response to its binding. The system should allow cloning of the gene using complementation of this phenotype in transfected cell lines.

Functional Evidence for Involvement of Multiple Putative Tumor Suppressor Genes on the Short Arm of Chromosome 3 in Human Oral Squamous Cell Carcinogenesis

Cytogenetic and restriction fragment length polymorphism (RFLP) analyses have suggested that a putative tumor suppressor genes(s), which may play an important role in the development of human oral squamous cell carcinoma (SCC), is located on the short arm of chromosome 3 (3p). We previously reported that introducing an intact human chromosome 3 into three different oral SCC tumorigenic cell lines completely suppresses the tumorigenicity of each cell line with significant decrease in the in vitro growth rate and morphological changes. To map the tumor suppressor gene(s) on 3p, we have now examined the tumorigenicity of microcell hybrid clones containing various fragments derived from 3p that were introduced by microcell-mediated chromosome transfer. Sixteen hybrid clones were obtained from four successful experiments, and these clones were classified into two groups: 4 fully tumorigenic clones and 12 suppressed phenotype clones. Analyses of the 3p segments in the series of hybrid clones with the use of RFLP or microsatellite markers revealed that the 3p21.2–p21.3 or 3p25 regions or both were consistently retained in the 12 clones with suppressed phenotype but not in the 4 tumorigenic clones. The more proximal 3p13 region also was retained in three nontumorigenic clones. The overall results are fairly compatible with recent evidence that there are three discrete regions on 3p showing frequent allelic losses on oral SCC, and they directly provide functional evidence for the presence of tumor-suppressor genes for oral SCC in these regions. The possibility that three genes, FHIT, VHL, and Tβ R-II, recently identified on 3p may be significantly involved in oral SCC development is also discussed.

Human mRNA Capping Enzyme (RNGTT) and Cap Methyltransferase (RNMT) Map to 6q16 and 18p11.22–p11.23, Respectively

Eukaryotic cellular and viral mRNAs contain a 59-terminal mGpppN cap structure, which has important consequences at multiple levels of gene expression including RNA splicing, 39 end formation, transcript stability, nuclear export, and translation initiation (6, 12, 15, 16). Recent studies have demonstrated that capping enzyme selectively binds to the phosphorylated, elongating form of RNA polymerase II (pol II) (23). This finding accounts for the specific capping of pol II transcripts. It may also provide new insights into how HIV tat protein promotes pol II phosphorylation by tat-activated kinase (TAK) to enhance polymerase processivity (5). Caps are formed on nascent pre-mRNAs by the sequential action of RNA 59-triphosphatase, which removes the g-phosphate of the initiating nucleotide, RNA guanylyltransferase, which transfers GMP from GTP to the resulting diphosphate end, and RNA (guanine-7) methyltransferase, which methylates the guanine N7 position of the newly formed GpppN termini (4, 12, 15). These three enzymatic activities are present as separate polypeptides in yeasts, and strains null for the triphosphatase (18), guanylyltransferase (14), or methyltransferase (7) are nonviable. Metazoans including humans contain a separate cap methyltransferase (RNA guanine-7-methyltransferase, RNMT), but the RNA 59-triphosphatase and guanylyltransferase activities are contained as Nand C-terminal domains in a bifunctional capping enzyme (RNGTT) encoded by a single gene (23). Despite this difference in genomic organization between uniand multicellular organisms, capping enzyme is functionally conserved, and Saccharomyces cerevisiae strains null for guanylyltransferase or RNA 59-triphosphatase are complemented for growth by the mammalian counterpart (21, 23). In light of the importance of mRNA capping for gene expression, we determined the chromosomal location of the human capping enzyme gene (RNGTT) by PCR using as template the NIGMS human/rodent somatic cell hybrid panel 2, version 3 (Coriell Institute for Medical Research Cell Repositories, Camden, NJ). This panel consists of DNA isolated from 24 human/rodent somatic cell hybrid lines that each retained one intact human chromosome. Primers 1, 59-CAGTGTGTCCATTCTGGCAGTGGTTTTG-39, and 2, 59GTGGGAGAGCATTTTCTGGTGACAGC-39, from the 39-UTR of the human capping enzyme cDNA sequence (Accession No. AF025654) were designed to give a human-specific 318-bp PCR product. PCR results demonstrated that the human capping enzyme gene RNGTT is located on chromosome 6 (data not shown). Fine-mapping was performed by PCR analysis of the GeneBridge 4 radiation hybrid panel (Research Genetics, Inc., Huntsville, AL) with 25 ng template genomic DNA and primers 1 and 2 in a 12.5-ml reaction. After an initial denaturation step at 94°C for 5 min, 35 cycles of amplification consisting of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s were performed, followed by a final extension of 5 min at 72°C. Amplification products were analyzed by agarose gel electrophoresis. Data were submitted to the Whitehead Institute/MIT Center for Genome Research STS mapping server for statistical analysis with the RHMAPPER software package (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl). The data vector for RNGTT was 00100 01101 02101 0011

Simultaneous Transfer of Mitochondrial DNA and Single Chromosomes in Somatic Cells: A Novel Approach for the Study of Defects in Nuclear-Mitochondrial Communication

The assembly and function of respiratory-competent mitochondria in eukaryotic cells depends on collaboration between the nuclear and mitochondrial genomes, but the molecular mechanisms underlying such cross-talk are poorly understood. Microcell-mediated chromosome transfer has been used to transfer intact chromosomes from one mammalian cell to another, helping to map loci implicated in different diseases and in the senescence process. In the present work, we show that microcells have a significant number of mitochondria which can be transferred to another cell simultaneously with a limited number of chromosomes. By fusing microcells from a colon carcinoma cell line with a mitochondrial DNA (mtDNA)-less osteosarcoma cell line, we were able to isolate transmitochondrial hybrids containing only one of three selectable chromosomes and mtDNA from the donor cell. The proportion of transmitochondrial hybrids containing one chromosomal marker with respect to the total transmitochondrial hybrids and cybrids was approximately 1% and no hybrids were isolated containing more than one nuclear marker. The genetic data correlated well with the composition and structure of the microcell preparations, which showed the presence of cytoplast-like structures and microcells containing mitochondria surrounding the micronuclei. Microcell-mediated mtDNA and chromosome transfer can be used to identify nuclear factors implicated in mtDNA maintenance and gene expression, as well as to investigate nuclear factors which modulate clinical phenotypes in mitochondrial disorders.

Establishing the Cryptosporidium parvum karyotype by NotI and SfiI restriction analysis and Southern hybridization.

The molecular karyotype of the coccidian parasite Cryptosporidium parvum has proven difficult to study because chromosomes of similar sizes migrate together when submitted to pulsed-field gel electrophoresis (PFGE). In the present work, the karyotype was studied by restriction of chromosome-sized DNA with the rare-cutting enzymes NotI and SfiI, followed by PFGE separation of the restriction fragments and Southern hybridization. These experiments showed that the C. parvum karyotype is formed by eight chromosomes, ranging in size from approximately 0.95 to 1.45 million base pairs (Mbp), accounting for a genome size of 9.6Mbp. As a first step towards the construction of a physical map of the C. parvum genome, a total of 20 probes, including 16 genes and the ribosomal DNA (rDNA) sequence, was mapped to intact chromosomes and to their restriction fragments. In this way, all chromosomes, but one, were identified by specific markers. A comparison of mapping data of homologous genes from different species belonging to the phylum Apicomplexa showed differences in the distribution of rDNA sequences and in the chromosomal localization of alpha- and beta-tubulin genes. The variation in genome size among these parasites is also discussed.

Mapping a novel cellular-senescence gene to human chromosome 2q37 by irradiation microcell-mediated chromosome transfer.

To identify the subchromosomal region that carries the cellular-senescence-restoring program of the human cervical carcinoma cell line SiHa, we constructed by irradiation microcell-mediated chromosome transfer a library of mouse A9 cells containing various fragments of human chromosome 2 tagged with pSV2neo in 2p11-p12. Eighty-seven clones were isolated and screened for the presence of human sequences by inter-Alu and inter-L1 polymerase chain reaction (PCR), and six clones exhibiting PCR-laddering patterns that differed from those of the A9 cells containing an intact chromosome 2 were examined further. Chromosome analysis and fluorescence in situ hybridization (FISH) using human-specific repetitive sequences revealed that four of these clones contained single subchromosomal transferable fragments (STFs). Southern blot hybridization of 14 cosmid markers revealed that the STFs in A9 cells were derived from human chromosome 2. These STFs were transferred into SiHa cells by microcell fusion, and one of the STFs restored the cellular-senescence program. The concordance of the cellular-senescence-restoring program with the presence or absence of specific DNA fragments of chromosome 2 indicated that the putative cellular-senescence gene was located in 2q32-qter. For more detailed mapping, we constructed mouse A9 cells containing STFs derived from human chromosome 2 tagged with pSTneo at different regions in 2q31-qter. PCR-laddering and FISH analyses were used to identify six clones that contained different STFs. These STFs were transferred into SiHa cells, and one of the three clones that restored cellular senescence contained a small fragment of human chromosome 2. This STF was shown by PCR analysis using 14 human chromosome 2-specific primer pairs to be smaller than 12.2 cM and was mapped to the 2q37 region by FISH analysis with inter-Alu PCR. Beta-galactosidase activity, which is a biomarker of senescent cells, and telomerase activity similar to that found in parental SiHa cells were detected in SiHa microcell hybrids, suggesting that the putative cellular-senescence gene was not involved in a telomerase pathway but rather in an alternate pathway of cellular senescence.

DNA topoisomerase II can drive changes in higher order chromosome architecture without enzymatically modifying DNA.

Topoisomerase II has been suggested to play a major role in chromosome organization based on its DNA decatenating activity and its ability to mediate direct binding interactions between DNA and nuclear matrix. However, this latter point remains controversial. Here we address the question of whether the chromatin binding activity of Topoisomerase II is sufficient to modify chromosome form using whole mammalian chromosomes in vitro. Intact chromosomes were microsurgically removed from living cells and disassembled by treatment with protease or heparin. When these disassembled chromosomes were incubated with recombinant human Topoisomerase II, the enzyme became incorporated into chromatin and reassembly resulted, leading to almost complete restoration of pre-existing chromosome shape and position within minutes. Chromosome reconstitution by Topoisomerase II was dose-dependent, saturable, and appeared to be controlled stoichiometrically, rather than enzymatically. Similar reassembly was observed in the absence of ATP and when a catalytically inactive thermosensitive Topoisomerase II mutant was used at the restrictive temperature. Chromosome recondensation also could be induced after the strand-passing activity of Topoisomerase II was blocked by treatment with an inhibitor of its catalytic activity, amsacrine. When a non-hydrolyzable beta,gamma-imido analog of ATP (AMP-PNP) was used to physiologically fix bound Topoisomerase II enzyme in a closed form around DNA, subsequent chromosome disassembly was prevented in the presence of high salt. These data suggest that Topoisomerase II may control higher order chromatin architecture through direct binding interactions, independently of its well-known catalytic activity.

Inducing the loss of conditionally dispensable chromosomes in Nectria haematococca during vegetative growth.

A procedure for inducing and detecting the loss of conditionally dispensable (CD) chromosomes in filamentous fungi during vegetative growth was developed using Nectria haematococca mating population VI as a model. CD chromosomes in two different isolates of N. haematococca were tagged via integrative transformation with a gene conferring resistance to hygromycin B. In each case the transformation vector included chromosome-specific DNA in order to direct its homologous recombination with the desired chromosome. Chromosome loss was induced by exposing tagged isolates to inhibitory concentrations of benomyl either for protracted periods of time on solid medium or for short periods of time in liquid medium. After exposure to benomyl, isolates that lost the tagged chromosome were identified by their loss of resistance to hygromycin B. Electrophoretic karyotyping was used to verify that isolates which failed to grow on hygromycin B lacked an intact CD chromosome. Ten other chemicals known to interfere with mitotic events or cell development in other organisms did not induce CD chromosome loss in N. haematococca.

Modification of bacterial artificial chromosomes through chi-stimulated homologous recombination and its application in zebrafish transgenesis.

The modification of yeast artificial chromosomes through homologous recombination has become a useful genetic tool for studying gene function and enhancer/promoter activity. However, it is difficult to purify intact yeast artificial chromosome DNA at a concentration sufficient for many applications. Bacterial artificial chromosomes (BACs) are vectors that can accommodate large DNA fragments and can easily be purified as plasmid DNA. We report herein a simple procedure for modifying BACs through homologous recombination using a targeting construct containing properly situated Chi sites. To demonstrate a usage for this technique, we modified BAC clones containing the zebrafish GATA-2 genomic locus by replacing the first coding exon with the green fluorescent protein (GFP) reporter gene. Molecular analyses confirmed that the modification occurred without additional deletions or rearrangements of the BACs. Microinjection demonstrated that GATA-2 expression patterns can be recapitulated in living zebrafish embryos by using these GFP-modified GATA-2 BACs. Embryos microinjected with the modified BAC clones were less mosaic and had improved GFP expression in hematopoietic progenitor cells compared with smaller plasmid constructs. The precise modification of BACs through Chi-stimulated homologous recombination should be useful for studying gene function and regulation in cultured cells or organisms where gene transfer is applicable.

Immunolocalization of actin in intact and DNA- and histone-depleted nuclei and chromosomes of allium cepa.

The presence of actin in eukaryotic nuclei and chromosomes, and especially in higher plant nuclei and chromosomes, has not been well established. We detected actin in meristematic cells of Allium cepa with indirect immunofluorescence technique and observed bright fluorescence in the intact nuclei and chromosomes, indicating that actin is present in the nuclei and chromosomes of the higher plant. We labeled sections of the meristematic cells of A. cepa with immunogold technique, gold particles were found over the whole nuclei and a number of gold particles were concentrated in condensed chromatin and nucleoli, confirming the results of the immunofluoresence observations. We treated the nuclei and chromosomes of A. cepa with DNase I and 2M NaCl and obtained DNA- and histone-depleted nuclei and chromosomes. Indirect immunofluorescence tests showed that the DNA- and histone-depleted nuclei and chromosomes reacted positively with the anti-actin antibodies. These results demonstrate that actin exists not only in intact nuclei and chromosomes, but also in DNA- and histone-depleted nuclei and chromosomes of the plant. In addition, our immuno-fluorescence tests indicate that tropomyosin is present in the nuclei and chromosomes of A. cepa.

A novel repressor of P element transposition in Drosophila melanogaster.

We have discovered, in an inbred line (Loua) of Drosophila melanogaster from Zaire, a third chromosome showing unusual P element repression. Repression of P element transposition by this chromosome, named Loua3, is dominant zygotic and has three unusual properties. Firstly, its repression of the gonadal dysgenesis caused by a strong P haplotype is strongly temperature-dependent, being most evident at higher rearing temperatures. Secondly, subdivision of Loua3 by recombination abolishes repression: the effect is apparently a function of the intact chromosome. Finally, Loua3 also diminishes somatic lethality when chromosomes carrying many 'ammunition' elements (Birmingham2) are exposed to the constitutive transposase source delta 2-3(99B). The chromosome has 17 P elements, none full-length, located in at least 12 dispersed positions.