A physical map for an Asian malaria mosquito, Anopheles stephensi

Physical mapping is a useful approach for studying genome organization and evolution as well as for genome sequence assembly. The availability of polytene chromosomes in malaria mosquitoes provides a unique opportunity to develop high-resolution physical maps. We report a 0.6-Mb-resolution physical map consisting of 422 DNA markers hybridized to 379 chromosomal sites of the Anopheles stephensi polytene chromosomes. This makes An. stephensi second only to Anopheles gambiae in density of a physical map among malaria mosquitoes. Three hundred sixty-three (363) probes hybridized to single chromosomal sites, whereas 59 clones yielded multiple signals. This physical map provided a suitable basis for comparative genomics, which was used for determining inversion breakpoints, duplications, and origin of novel genes across species.     

Low-resolution genome map of the malaria mosquito Anopheles gambiae.

We have microdissected divisions of the Anopheles gambiae polytene chromosomes, digested the DNAs with a restriction enzyme, and PCR-amplified the DNA fragments to generate a set of pooled probes, each corresponding to approximately 2% of the mosquito genome. These divisional probes were shown to have high complexity. Except for those derived from near the centromeres, they hybridize specifically with their chromosomal sites of origin. Thus, they can be used to map cloned DNAs by a dot blot procedure, which is much more convenient than in situ hybridization to polytene chromosomes. We discuss additional potential uses of these probes, such as easier isolation of molecular markers and genes, including those that cross-hybridize with clones available from other insects. It is expected that the probes will substantially accelerate molecular genetic analysis of this most important malaria vector.     

Butylhydroxytoluene (BHT) increases susceptibility of transgenic rasH2 mice to lung carcinogenesis

PURPOSE: Transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mice) are highly susceptible to lung carcinogens. In order to investigate the possibility of developing a rapid in vivo assay for lung carcinogens, we examined whether the tumor-promoting activity of butylhydroxytoluene (BHT) is efficacious in rasH2 mice. METHODS: rasH2 mice and wild littermates of both genders were pre-treated with carcinogens [urethane (UR), 4-nitroquinoline 1-oxide (4NQO) or diethylnitrosamine (DEN)], and, one day later, given a 400 mg/kg dose of BHT. RESULTS: Six weeks after the initiation treatment, evidence of carcinogenicity could be detected in male and female rasH2 mice that had received UR doses of > or = 250 mg/kg and > or = 125 mg/kg, respectively, prior to exposure to BHT, whereas only 500 mg/kg of UR was sufficient to induce tumors in female rasH2 mice given the carcinogen alone. The carcinogenicity of 15 mg/kg of 4NQO could be detected after 9 weeks in male rasH2 mice given the carcinogen followed by BHT. Similarly, the carcinogenicity of 60 mg/kg of DEN could be detected after 9 weeks and 6 weeks, respectively, in male and female rasH2 mice given the carcinogen followed by BHT. No carcinogenicity could be demonstrated through the experimental period with doses of 4NQO or DEN given alone. CONCLUSIONS: These results indicate that BHT administration increases the susceptibility of rasH2 mice to lung carcinogens, and suggest that the use of BHT in rasH2 mice might lead to the establishment of a rapid in vivo assay for lung carcinogens.     

DNA transposon Hermes inserts into DNA in nucleosome-free regions in vivo

Transposons are mobile genetic elements that are an important source of genetic variation and are useful tools for genome engineering, mutagenesis screens, and vectors for transgenesis including gene therapy. We have used second-generation sequencing to analyze ≈2 × 10(5) unique de novo transposon insertion sites of the transposon Hermes in the Saccharomyces cerevisiae genome from both in vitro transposition reactions by using purified yeast genomic DNA, to better characterize intrinsic sequence specificity, and sites recovered from in vivo transposition events, to characterize the effect of intracellular factors such as chromatin on target site selection. We find that Hermes transposon targeting in vivo is profoundly affected by chromatin structure: The subset of genome-wide target sites used in vivo is strongly associated (P < 2e-16 by Fisher's exact test) with nucleosome-free chromatin. Our characterization of the insertion site preferences of Hermes not only assists in the future use of this transposon as a molecular biology tool but also establishes methods to more fully determine targeting mechanisms of other transposons. We have also discovered a long-range sequence motif that defines S. cerevisiae nucleosome-free regions.     

High-resolution analysis of Drosophila heterochromatin organization using SuUR Su(var)3-9 double mutants

The structural and functional analyses of heterochromatin are essential to understanding how heterochromatic genes are regulated and how centromeric chromatin is formed. Because the repetitive nature of heterochromatin hampers its genome analysis, new approaches need to be developed. Here, we describe how, in double mutants for Su(var)3-9 and SuUR genes encoding two structural proteins of heterochromatin, new banded heterochromatic segments appear in all polytene chromosomes due to the strong suppression of under-replication in pericentric regions. FISH on salivary gland polytene chromosomes from these double mutant larvae allows high resolution of heterochromatin mapping. In addition, immunostaining experiments with a set of antibodies against euchromatic and heterochromatic proteins reveal their unusual combinations in the newly appeared segments: binding patterns for HP1 and HP2 are coincident, but both are distinct from H3diMetK9 and H4triMetK20. In several regions, partial overlapping staining is observed for the proteins characteristic of active chromatin RNA Pol II, H3triMetK4, Z4, and JIL1, the boundary protein BEAF, and the heterochromatin-enriched proteins HP1, HP2, and SU(VAR)3-7. The exact cytological position of the centromere of chromosome 3 was visualized on salivary gland polytene chromosomes by using the centromeric dodeca satellite and the centromeric protein CID. This region is enriched in H3diMetK9 and H4triMetK20 but is devoid of other proteins analyzed.     

Structural and functional differences between histone H1 sequence variants with differential intranuclear distribution.

The chromatin of most cell types contains several different sequence variants of histone H1. The functional role of this heterogeneity is not known. In the larval tissues of the midge, Chironomus thummi, there are H1 variants of two types. H1 II-1, H1 II-2, and H1 III-1 have similar amino acid sequences and appear uniformly distributed in polytene interphase chromosomes. The total number of gene copies per genome for this type of H1 histones is about 40 in C. th. thummi and 50-60 in C. th. piger. In contrast, histone H1 I-1 is encoded by a single copy gene in C. th. thummi and by two to four genes in C. th. piger. It has a divergent structure and is found only in a limited number of condensed chromosome sites. The N-terminal domain of H1 I-1 contains an insertion that is lacking in the other H1 variants and that is part of a variant-specific bipartite sequence Lys-Ala-Pro-Lys-Ala-Pro-Xaa10-Lys-Val-Ala in front of the conserved central domain. N-terminal peptides of H1 I-1 including this motif, in contrast to the homologous peptide from H1 II-1, competed with the drug Hoechst 33258 for binding to the minor groove of the DNA double helix. Repeats of the sequence Lys-Ala-Pro are also present at the same distance from the conserved central domain, in a single H1 variant of a nematode and of a green alga. The motif could interact with linker DNA in intranuclear targeting or packaging a condensed subtype of chromatin, or both.     

Detection of exocyclic 1,N2-propanodeoxyguanosine adducts as common DNA lesions in rodents and humans.

Exocyclic adducts are unique DNA modifications resulting from binding at two sites of bases that normally are involved in hydrogen-bonding for maintaining the double-helical structure of DNA. These adducts have been shown to be formed in rodents upon exposure to carcinogens. Using a sensitive 32P-postlabeling method combined with high performance liquid chromatography, we obtained evidence that 1,N2-propanodeoxyguanosine adducts of acrolein (AdG) and crotonaldehyde (CdG) are present in the liver DNA of humans and rodents without carcinogen treatment. The identities of these adducts were verified by cochromatography with the synthetic adduct standards. Further proof of identities was obtained by conversion mediated by nuclease P1 of the labeled AdG and CdG 3',5'-bisphosphates to their corresponding 5'-monophosphates. This treatment converted the in vivo adducts into products that again cochromatographed in a characteristic pattern with the synthetic 5'-monophosphates of AdG and CdG. Using this assay, we also demonstrated the in vivo stereoselective formation of one of the AdG isomers. The estimated total levels of modification were 1.0-1.7, 0.2-1.0, and 0.3-2.0 adducts in 10(6) guanine bases in the liver DNA of mice, rats, and humans, respectively. The detection of these adducts in relatively high levels without carcinogen treatment suggests that the endogenous factors such as lipid peroxidation may be important for their formation. This study provides evidence for the presence of acrolein- and crotonaldehyde-derived exocyclic adducts as common lesions in the liver DNA of rodents and humans.     

High yield of micronuclei and micronuclei premature chromosome condensation in a mouse tumor cell line cultured in vivo with prearrested mitotic metaphases

It is generally argued that micronuclei and micronuclei premature chromosome condensation (MN PCC) in stable cell lines occur only after treatment with potent clastogens, mutagens or carcinogens. In the present paper we report on the occurrence of micronuclei and MN PCC (predominantly of S type) at a high frequency in a mixed in vivo culture of prearrested mitotic metaphases with an asynchronous population of mouse ascites tumor cells (S180) without the aid of any clastogen, mutagen or carcinogen.     

A spontaneously opened ring chromosome of Drosophila melanogaster has acquired He-T DNA sequences at both new telomeres.

Ring chromosomes that have been opened to give linear chromosomes offer an opportunity to study the DNA sequences associated with new chromosome ends. The Drosophila melanogaster chromosome C(1)A was originally a ring chromosome, consisting of two linked X chromosomes, and thus had no telomeres. This chromosome has spontaneously opened in polytene region 13, a region near the middle of the euchromatic arm of the X chromosome. The opening of the ring has produced two new telomeres on the C(1)A chromosome. Each of the new telomeres has acquired He-T DNA sequences. He-T DNA is a complex family of repeated sequences found in the telomeric and pericentric heterochromatin of D. melanogaster chromosomes. He-T DNA sequences are detected, at various levels, in the most distal band on the end of each polytene chromosome in all D. melanogaster stocks. To our knowledge, these sequences have never been detected within the euchromatic chromosomal regions in any stock. The strong correlation between He-T DNA sequences and telomeric regions suggests that He-T sequences may have a role in organizing or maintaining the ends of chromosomes. The association of He-T DNA with newly acquired telomeres in a formerly euchromatic region, polytene region 13, strengthens this correlation.     

五条蚋两地理株多线染色体比较研究

【提要】 对五条蚋贵州株和江西株多线染色体进行描述和比较，并绘制模式图。分别取成熟幼虫分离出唾液腺，经苯酚品红染液染色、压片，镜检、摄影和测量，作统计学处理。发现两地理株多线染色体数目为3条，主要特征性结构高度一致，唯ⅡL带型分布有显著差异。     

A multimember kinesin gene family in Drosophila.

Degenerate primers to the kinesin motor domain were used in the polymerase chain reaction to amplify DNA sequences from Drosophila genomic DNA and cDNA libraries. The amplified DNA sequences were hybridized to polytene chromosomes and the map positions of the hybridizing sites were determined. More than 30 sites of hybridization were detected, indicating that the kinesin gene family may be much larger than previously thought. One new family member has already been identified as a result of this screen. The map positions should aid in the identification of further kinesin family members. Some of these kinesin-related genes are anticipated to function in previously undiscovered roles in the cell.     

Drosophila histone H2A.2 is associated with the interbands of polytene chromosomes.

Drosophila chromatin contains two antigenically distinct H2A histones, H2A.1 and H2A.2. Indirect immunofluorescence analyses revealed that anti-H2A.1 binding was distributed throughout polytene chromosomes, whereas anti-H2A.2 binding was interband-specific. Thus, H2A.2 probably contributes to the less compacted structure of interbands. Since each band-interband region is thought to contain a single gene, our results suggest that the distribution of H2A.2 echoes the functional organization of the Drosophila genome. Similar H2A histones occur in eukaryotes ranging from protozoa to mammals. Their placement might be an important determinant of chromatin structure.     

Does heterochromatin protein 1 always follow code?

Heterochromatin protein 1 (HP1) is a conserved chromosomal protein that participates in chromatin packaging and gene silencing. A loss of HP1 leads to lethality in Drosophila and correlates with metastasis in human breast cancer cells. On Drosophila polytene chromosomes HP1 is localized to centric regions, telomeric regions, in a banded pattern along the fourth chromosome, and at many sites scattered throughout the euchromatic arms. Recently, one mechanism of HP1 chromosome association was revealed; the amino-terminal chromo domain of HP1 interacts with methylated lysine nine of histone H3, consistent with the histone code hypothesis. Compelling data support this mechanism of HP1 association at centric regions. Is this the only mechanism by which HP1 associates with chromosomes? Interest is now shifting toward the role of HP1 within euchromatic domains. Accumulating evidence in Drosophila and mammals suggests that HP1 associates with chromosomes through interactions with nonhistone chromosomal proteins at locations other than centric heterochromatin. Does HP1 play a similar role in chromatin packaging and gene regulation at these sites as it does in centric heterochromatin? Does HP1 associate with the same proteins at these sites as it does in centric heterochromatin? A first step toward answering these questions is the identification of sequences associated with HP1 within euchromatic domains. Such sequences are likely to include HP1 "target genes" whose discovery will aid in our understanding of HP1 lethality in Drosophila and metastasis of breast cancer cells.     

Granules 25-30 nm in diameter: basic constituent of the nuclear matrix, chromosome scaffold, and nuclear envelope.

Rat liver nuclear matrix and similar structures derived from isolated Chironomus polytene chromosomes, nuclear envelopes, and intranuclear bodies of frog late oocytes (the karyospheres) were studied by electron microscopy with platinum shadowing and negative staining. We have shown that the treatment of whole nuclei, nuclear envelopes, polytene chromosomes, or karyospheres with nonionic detergent, high salt, and RNase and DNase followed by dilute alkali or hyaluronidase digestion reveals numerous rather uniform granules 25-30 nm in diameter. With omission of the nucleases the granules appear to be associated with DNA strands mostly organized in loops. Many granules form clusters and are arranged in linear or arch-like aggregates or cycles resembling the pore complexes. We suppose that these spherical bodies constitute a basic component of the nuclear matrix, chromosome scaffold, and nuclear envelope and are bound together by hyaluronic acid or some similar glycosaminoglycan.