Homologous banding patterns in the polytene chromosomes from the larval salivary glands and ovarian nurse cells of Anopheles stephensi liston (Culicidae)

A comparison of the banding patterns of two homologous polytene chromosome arms from the larval salivary gland and ovarian nurse cell complement of Anopheles stephensi is presented. The homologous chromosomes from the somatic larval salivary glands and germ-line derived ovarian nurse cells have essentially the same band-interband organisation. An analysis of the ³H-uridine labelling patterns of a small chromosome segment from the two tissues indicates that germ-line polytene chromosomes are not radically different from somatic polytene chromosomes in their patterns of gene expression.     

Satellite DNA of Anopheles stephensi liston (Diptera: Culicidae)

Four satellite DNAs in the Anopheles stephensi genome have been defined on the basis of their banding properties in Hoechst 33258-CsCl density gradients. Two of these satellites, satellites I and II, are visible on neutral CsCl density gradients as a light density peak forming approximately 15% of total cellular DNA. Hoechst-CsCl density gradient profiles of DNA extracted from polytene tissues indicates that these satellites are underreplicated in larval salivary gland cells and adult female Malpighian tubules and possibly also in ovarian nurse cells. The chromosomal location of satellite I on mitotic and polytene chromosomes has been determined by in situ hybridisation. Sequences complementary to satellite I are present in approximately equal amounts on a heterochromatic arm of the X and Y chromosomes and are also present, in smaller amounts, at the centromere of chromosome 3. A quantitative analysis of the in situ hybridisation experiments indicates that sequences complementary to satellite I at these two sites differ in their replicative behaviour during polytenisation: heterosomal satellite I sequences are under-replicated relative to chromosome 3 sequences in polytene larval salivary gland and ovarian nurse cell nuclei.     

Underreplication of satellite DNAs in polyploid ovarian tissue of Drosophila virilis

The satellite DNAs of Drosophila virilis have been examined in diploid and polyploid tissues by isopycnic ultracentrifugation and thermal denaturation experiments. Previous work has established that the satellite DNAs are under replicated in the polytene chromosomes of the salivary glands of D. virilis. The results of the present experiments demonstrate that this underreplication also takes place in the ovaries which contain nurse cells and follicle cells. These tissues are polyploid but do not show polytene chromosomes.     

普通小麦7B染色体两类低拷贝专化DNA序列的特性

研究表明 ,多倍体小麦基因组中存在一类低拷贝、染色体专化的DNA序列 ,其在多倍体形成时常表现出不稳定性。这类序列被认为在异源多倍体的建立和稳定中起着关键作用。为进一步研究这一问题 ,对通过染色体显微切割从普通小麦 (TriticumaestivumL .)中分离的 5个 7B染色体专化DNA序列的特性进行了研究。以这些序列为探针对大量的多倍体小麦和它们的二倍体祖先物种进行了Southern杂交分析。结果表明 ,这些序列可被分为两种类型 :其中的 4个序列与所有的多倍体物种均杂交 ,但是在二倍体水平上 ,它们却只与和多倍体小麦B基因组紧密相关的物种杂交 ,这说明这些序列是在二倍体物种分化以后产生的 ,然后垂直传递给多倍体 ;其中的 1个序列与所有的二倍体及多倍体物种均杂交 ,暗示在多倍体形成后这些序列从A和D基因组中消除了。用这一序列分别与一个人工合成的六倍体和四倍体小麦进行Southern杂交的结果表明 ,序列消除是一个迅速的事件而且很可能与这些序列的甲基化状态有关。认为这些低拷贝的染色体专化序列对于多倍体形成后部分同源染色体之间的进一步分化起着重要作用。     

Automictic parthenogenesis in the stick insectBacillus rossius Rossi (Cheleutoptera,phasmidae)

The process which restores the zygoid number of chromosomes in the thelytokously parthenogenetic stick insectBacillus rossius Rossi, is described. During the first 15 days after oviposition approximately 200 haploid (A+X=18 chromosomes) pre-blastoderm cells develop in the egg-cortex. During the second period of 15 days up to about 2000 blastoderm cells develop in which were found: haploid mitoses, C-mitoses (A+X=18 chromosomes), endomitoses, diploid mitoses (2A+2X=36 chromosomes) and pycnosis. The next 10 days the number of pycnotic nuclei decreases gradually and the blastoderm cells multiply by diploid mitosis only. Thereafter, a diploid germ band is formed either directly or after a diapause. It is concluded that C-mitosis in haploid cells restores the somatic number of chromosomes (automictic parthenogenesis), that a portion of the cells in C-mitosis degenerates and that endomitosis leads to the formation of some polyploid cells only. This development is discussed in relation to the occurrence of geographic parthenogenesis and of impaternate males.     

Under-replication of intron+ rDNA cistrons in polyploid nurse cell nuclei of Calliphora erythrocephala

We have examined the possibility that the intron-containing (intron⁺) rDNA cistrons of Dipteran flies are active in the germ-line derived polyploid nurse cell nuclei of the ovarian follicles. Using the organism, Calliphora erythrocephala, we describe here a procedure which yields very pure nurse cell nuclei and compare the intron-free (intron^–) and intron⁺ rDNA cistron contents of nurse cell nuclei prepared by this procedure to those of 2–18 h embryo nuclei and 3 day pupal nuclei. DNA from three preparations of each nuclear type was examined and the intron^– and intron⁺ cistron contents quantitated using a Southern transfer procedure. The number of intron^– and intron⁺ rDNA cistrons per haploid genome in the presumed diploid 2–18 h embryo DNA was first established, and then the intron^– and intron⁺ cistron contents of nurse cell nuclear DNA and 3 day pupal DNA were determined relative to these values.The intron^– cistron content of nurse cell nuclear DNA was indistinguishable from that of embryonic DNA but the intron⁺ cistrons showed an 8-fold under-replication relative to the presumed diploid DNA. A slight under-representation of the intron^– cistrons and 3-fold under-replication of the intron⁺ cistrons were demonstrated for 3 day pupal DNA. These findings strongly suggest that intron⁺ rDNA cistrons are non-functional in nurse cell nuclei and substantiate the generality of this implication for the whole organism during early pupal life.     

The replicative organization of DNA in polytene chromosomes ofDrosophila hydei

Salivary-gland nuclei ofDrosophila hydei were pulse-labeledin vitro with³H-thymidine and studied autoradiographically in squash preparations. The distribution of radioactive label over the length of the polytene chromosomes was discontinuous in most of the labeled nuclei; in some nuclei the pattern of incorporation was continuous. Comparison of the various labeling patterns of homologous chromosome regions in different nuclei showed that specific replicating units are replicated in a specific order. By combining autoradiography with cytophotometry of Feulgen-stained chromosomes, it was possible to correlate thymidine labeling of specific bands with their DNA content. The resulting data indicate that during the S-period many or perhaps all of the replicating units in a salivary-gland nucleus start DNA synthesis simultaneously but complete it at different times. Furthermore, the data support the hypothesis that the chromomere is a unit of replication or replicon. The DNA content of haploid chromomeres was found to be about 5×10^-4 pg for the largest bands inDrosophila hydei. From the results of H³-thymidine autoradiography and Feulgen-cytophotometry on neuroblast and anlage nuclei it was concluded that during growth of the polytenic nucleus heterochromatin is for the most part excluded from duplication. The results of DNA measurements in interbands of polytene chromosomes do not agree with a multistrand structure for the haploid chromatid. A chromosome model is proposed which is in accordance with the reported results and with current views concerning the replicative organization of chromosomes.     

A novel transposon-like repeat interrupted by an LTR element occurs in a cluster of B1 repeats in the mouse c-mos locus.

The mouse genomic locus containing the oncogene c-mos was analyzed for repetitive DNA sequences. We found a single B1 repeat 10 kb upstream and three B1 repeats 0.6 kb, 2.7 kb, and 5.4 kb, respectively, downstream from c-mos. The B1 repeat closest to c-mos contains an internal 7-bp duplication and a 18-bp insertion. Localized between the last two B1 repeats is a copy of a novel mouse repeat. Sequence comparison of three copies of this novel repeat family shows that they a) contain a conserved BglII site, b) are approximately 420 bp long, c) possess internal 50-bp polypurine tracts, and d) have structural characteristics of transposable elements. They are present in about 1500 copies per haploid genome in the mouse, but are not detectable in DNA of other mammals. The BglII repeat downstream from c-mos is interrupted by a single 632-bp LTR element. We estimate that approximately 1200 copies of this element are present per haploid genome in BALB/c mice. It shares sequence homology in the R-U5 region with an LTR element found in 129/J mice.     

Ectopic expression of the Suppressor of Underreplication gene inhibits endocycles but not the mitotic cell cycle in Drosophila melanogaster

The Suppressor of Underreplication ( SuUR) gene contributes to the regulation of DNA replication in regions of intercalary heterochromatin in salivary gland polytene chromosomes. In the SuUR mutant these regions complete replication earlier than in wild type and, as a consequence, undergo full polytenization. Here we describe the effects of ectopic expression of SuUR using the GAL4-UAS system. We demonstrate that ectopically expressed SuUR exerts qualitatively distinct influences on polyploid and diploid tissues. Ectopic expression of SuUR inhibits DNA replication in polytene salivary gland nuclei, and reduces the degree of amplification of chorion protein genes that occurs in the follicle cell lineage. Effects caused by ectopic SuUR in diploid tissues vary considerably; there is no obvious effect on eye formation, but apoptosis is observed in the wing disc, and wing shape is distorted. The effect of ectopic SuUR expression is enhanced by mutations in the genes E2F and mus209 ( PCNA). Differential responses of polyploid and diploid cells to ectopic SuUR may reflect differences in the mechanisms underlying mitotic cell cycles and endocycles.     

Characterization of a family of repetitive sequences that is eliminated late during macronuclear development of the hypotrichous ciliate Stylonychia lemnae

A repetitive element from the hypotrichous ciliate Stylonychia lemnae was characterized by restriction and hybridization analysis. This repetitive element is present in about 5,000–7,000 copies per haploid genome in the micronucleus and the macronuclear anlagen. Its DNA sequence is very conserved, but the length of the repetitive sequence blocs is variable. In some cases, it is associated with telomeric sequences and macronucleus–homologous sequences. Restriction analysis of genomic micronuclear and macronuclear anlagen DNA and in situ hybridization showed that the repetitive sequences are amplified during the formation of polytene chromosomes. They are localized in many bands of the polytene chromosomes and are eliminated during the degradation of the polytene chromosomes. Possible functions of the repetitive sequences during macronuclear differentiation are discussed. Dev. Genet. 21:201–211, 1997.© 1997 Wiley-Liss, Inc.     

Both Nucleolar Organizers Are Replicated in Dipteran Polyploid Tissues: A Study at the Level of Individual Nuclei

Working with the Dipteran Calliphora erythrocephala, we have tested the hypothesis that only one nucleolar organizer region (NO) is replicated during polyploidization. NO replication was examined in two very different highly polyploid nuclear types: salivary gland nuclei and nurse cell nuclei. Two strains of the organism containing NO regions with highly diagnostic nontranscribed spacer (NTS) polymorphisms were prepared and reciprocal single pair-matings between members of the strains were performed. The representation of the two distinguishable NOs in diploid and polyploid DNAs of individual F1 progeny from each cross was then examined. DNA from a total polyploid nuclear DNA preparation and from individual polyploid nuclei of both tissue types was analyzed. Our results show conclusively that both genomic NOs are replicated in individual polyploid nuclei of both types. Further, evidence for variation in the relative replication of cistrons from the two NOs by individual nuclei was obtained. The cistron types present in the NOs of both strains showed differential replication upon polyploidization. In general, the patterns of differential cistron replication seen in salivary gland and nurse cell nuclei were similar.     

Rapid Elimination of Low-Copy DNA Sequences in Polyploid Wheat: A Possible Mechanism for Differentiation of Homoeologous Chromosomes

To study genome evolution in allopolyploid plants, we analyzed polyploid wheats and their diploid progenitors for the occurrence of 16 low-copy chromosome- or genome-specific sequences isolated from hexaploid wheat. Based on their occurrence in the diploid species, we classified the sequences into two groups: group I, found in only one of the three diploid progenitors of hexaploid wheat, and group II, found in all three diploid progenitors. The absence of group II sequences from one genome of tetraploid wheat and from two genomes of hexaploid wheat indicates their specific elimination from these genomes at the polyploid level. Analysis of a newly synthesized amphiploid, having a genomic constitution analogous to that of hexaploid wheat, revealed a pattern of sequence elimination similar to the one found in hexaploid wheat. Apparently, speciation through allopolyploidy is accompanied by a rapid, nonrandom elimination of specific, low-copy, probably noncoding DNA sequences at the early stages of allopolyploidization, resulting in further divergence of homoeologous chromosomes (partially homologous chromosomes of different genomes carrying the same order of gene loci). We suggest that such genomic changes may provide the physical basis for the diploid-like meiotic behavior of polyploid wheat.     

Microdissection and sequence analysis of pericentric heterochromatin from the Drosophila melanogastermutant Suppressor of Underreplication

In the Suppressor of Underreplication( SuUR) mutant strain of Drosophila melanogaster, the heterochromatin of polytene chromosomes is not underreplicated and, as a consequence, a number of beta-heterochromatic regions acquire a banded structure. The chromocenter does not form in these polytene chromosomes, and heterochromatic regions, normally part of the chromocenter, become accessible to cytological analysis. We generated four genomic DNA libraries from specific heterochromatic regions by microdissection of polytene chromosomes. In situ hybridization of individual libraries onto SuUR polytene chromosomes shows that repetitive DNA sequences spread into the neighboring euchromatic regions. This observation allows the localization of eu-heterochromatin transition zones on polytene chromosomes. We find that genomic scaffolds from the eu-heterochromatin transition zones are enriched in repetitive DNA sequences homologous to those flanking the suppressor of forked gene [ su(f) repeat]. We isolated and sequenced about 300 clones from the heterochromatic DNA libraries obtained. Most of the clones contain repetitive DNA sequences; however, some of the clones have unique DNA sequences shared with parts of unmapped genomic scaffolds. Hybridization of these clones onto SuUR polytene chromosomes allowed us to assign the cytological localizations of the corresponding genomic scaffolds within heterochromatin. Our results demonstrate that the SuUR mutant renders possible the mapping of heterochromatic scaffolds on polytene chromosomes.     

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster

In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with ³H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S+18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S+18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. — In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.     

Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n?=?12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.     

Die Verteilung der Chromosomen auf die Tochterzellkerne multipolarer Mitosen in euploiden Gewebekulturen von Microtus agrestis

In kidney epithelial cultures from female Microtus agrestis, 3,55% of all mitoses are multipolar, 94% of them tripolar. Feulgen photometric measurements of 21 tripolar mitoses reveal a total DNA amount corresponding to the mitotic diploid value (4c) in 5 cases, and to the tetraploid value (8c) in 16 cases, Diploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei each with a haploid DNA value (1c). Most tetraploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei with a triploid DNA value (3c). Also the sex chromosomes are distributed to the daughter nuclei in the relation of 2∶3∶3. This can be seen in anaphase figures as well as in interphase nuclei presumably derived from tripolar mitoses, showing chromocenters according to the number of X-chromosomes. In two cases of tripolar tetraploid mitoses the resulting nuclei have a haploid, a triploid and a tetraploid DNA value. The DNA replication pattern is always identical in the daughter nuclei of diploid and tetraploid tripolar mitoses. — Our observations suggest segregation and distribution of haploid chromosome sets or multiples of haploid sets to the daughter nuclei of multipolar mitoses. They also show a possible way of formation of haploid and triploid cells in a basically diploid tissue. Presumably triploid nuclei (with 3 chromocenters) are capable of DNA synthesis.     

Cytological studies on the organization of DNA in giant trophoblast nuclei of the mouse and the rat

Giant trophoblast nuclei of the mouse and the rat, known to contain hundreds, or even thousands, of times the haploid amount of DNA, have been studied by a number of cytological techniques. These nuclei appear in two morphological states:“reticulate,” in which large numbers of chromatin threads of uniform size intermingle throughout the nucleus, often radiating from clumps of heterochromatin adjacent to the nucleoli, and “homogeneous,” in which the chromatin is more evenly dispersed and individual threads are more difficult to distinguish. Intermediate morphologies are also observed. In neither case were structures resembling polytene chromosomes discernible. — Centromeric heterohromatin as revealed by the Giemsa BSG technique has been quantitatively analyzed in giant versus diploid trophoblast nuclei. Although the median number of chromocenters is slightly greater in giant than in diploid nuclei, the range is similar. In both cases, the chromocenter number is usually less than the diploid number of chromosome pairs, indicating the attraction between centromeres not only of homologous, but also of heterologous, chromosomes. By scanning microdensitometry, we have observed a constant ratio of chromocenter area: total nuclear area in giant cells. This ratio, which likely reflects the ratio of chromocenter volume: total nuclear volume, is in good agreement with that of satellite DNA: total DNA.     

The African trypanosome genome

The haploid nuclear genome of the African trypanosome, Trypanosoma brucei, is about 35 Mb and varies in size among different trypanosome isolates by as much as 25%. The nuclear DNA of this diploid organism is distributed among three size classes of chromosomes: the megabase chromosomes of which there are at least 11 pairs ranging from 1 Mb to more than 6 Mb (numbered I-XI from smallest to largest); several intermediate chromosomes of 200-900 kb and uncertain ploidy; and about 100 linear minichromosomes of 50-150 kb. Size differences of as much as four-fold can occur, both between the two homologues of a megabase chromosome pair in a specific trypanosome isolate and among chromosome pairs in different isolates. The genomic DNA sequences determined to date indicated that about 50% of the genome is coding sequence. The chromosomal telomeres possess TTAGGG repeats and many, if not all, of the telomeres of the megabase and intermediate chromosomes are linked to expression sites for genes encoding variant surface glycoproteins (VSGs). The minichromosomes serve as repositories for VSG genes since some but not all of their telomeres are linked to unexpressed VSG genes. A gene discovery program, based on sequencing the ends of cloned genomic DNA fragments, has generated more than 20 Mb of discontinuous single-pass genomic sequence data during the past year, and the complete sequences of chromosomes I and II (about 1 Mb each) in T. brucei GUTat 10.1 are currently being determined. It is anticipated that the entire genomic sequence of this organism will be known in a few years. Analysis of a test microarray of 400 cDNAs and small random genomic DNA fragments probed with RNAs from two developmental stages of T. brucei demonstrates that the microarray technology can be used to identify batteries of genes differentially expressed during the various life cycle stages of this parasite.     

Organization of rabbit immunoglobulin genes. I. Structure and multiplicity of germ-line VH genes

Two rabbit germ-line VH gene segments have been isolated from a recombinant phage DNA library. Nucleotide sequence analysis indicates that both of the genes share structural and regulatory features common to mouse and human VH genes, although one appears to be a pseudogene. Comparison of the protein sequences encoded by these genes to the protein sequences of rabbit immunoglobulin V regions indicates that both genes encode VH a-negative-like molecules. Quantitative genomic blot analysis with a VH probe capable of recognizing most, if not all, germ-line VH genes indicates that there are approximately 100 VH genes in the haploid genome of rabbits. The average spacing between the germ-line VH genes was determined to be approximately 6 kb. The molecular basis for the allelic inheritance of rabbit VH allotypes is discussed in view of the structural organization of germ-line VH genes.