Showing papers on "Polytene chromosome published in 1985"
TL;DR: DNA topoisomerase II has been immunochemically identified on protein blots as a major polypeptide component of the Drosophila nuclear matrix-pore complex-lamina fraction and upon nuclear disassembly during mitosis appears to redistribute diffusely throughout the cell.
Abstract: DNA topoisomerase II has been immunochemically identified on protein blots as a major polypeptide component of the Drosophila nuclear matrix-pore complex-lamina fraction. Indirect immunofluorescence analyses of larval cryosections have confirmed the nuclear localization of topoisomerase II in situ. Although apparently excluded from the nucleolus, the topoisomerase protein is otherwise distributed throughout the interior of interphase nuclei. Similar immunocytochemical studies performed with permeabilized whole giant cells from third-instar larval salivary glands have shown topoisomerase II to be largely restricted to the polytene chromosomes. Upon nuclear disassembly during mitosis, the topoisomerase polypeptide appears to redistribute diffusely throughout the cell. Faint immunofluorescent staining of mitotic chromosomes is also observed.
435 citations
TL;DR: 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.
Abstract: 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.
106 citations
TL;DR: A transformed strain is recovered that carries an ry+ transposon inserted in or near the scalloped locus in polytene section 13F on the X chromosome, and this allele has been destabilized by subsequent P-M hybrid dysgenesis, and mutations were recovered that exhibit alterations in the rosy and/or scaloped phenotypes.
Abstract: Utilizing the method of P-M hybrid dysgenesis-mediated gene transfer to insert rosy locus DNA into various chromosomal locations, we recovered a transformed strain that carries an ry + transposon inserted in or near the scalloped locus in polytene section 13F on the Χ chromosome The resultant product, when stabilized, behaves as a homozygous and hemizygous viable and fertile extreme scalloped allele associated with wild-type expression of the rosy locus We have labeled this allele, sd ry + + This allele has been destabilized by subsequent P-M hybrid dysgenesis, and mutations were recovered that exhibit alterations in the rosy and/or scalloped phenotypes Representative samples of all phenotypic classes have been characterized by Southern blot analyses of restricted DNA The most common events are excisions of DNA wholly internal to the transposon and representing sections of rosy DNA In addition to loss of rosy locus function, such excisions affect the scalloped locus expression—A second dysgenesis experiment was carried out involving an ry + transposon inserted in polytene section 16D on the Χ chromosome A minimal estimate of the relative frequency of imprecise excisions, determined in this experiment is 75%—A successful pilot experiment is described that utilizes dysgenic perturbation of the sd ryry + allele to select for small deletions of the 59 noncoding region of the rosy locus
103 citations
TL;DR: The sequence further strengthens the hypothesis that the chymotrypsin cleavage specificity developed separately in the vertebrates and invertebrates.
Abstract: We have isolated a clustered gene family in D. melanogaster that codes for trypsin-like enzymes. The gene family has been localized to 47D-F by in situ hybridization to polytene chromosomes. The four genes in the family are transcribed in alternating orientations, and code for 1000 nt mRNAs. Transcripts are present at all stages of the life cycle. In situ hybridization to mRNA in tissue sections of third instar larvae showed that transcripts were restricted to the mid-gut. One gene was sequenced. The translated amino acid sequence of the proposed active enzyme is 42% homologous to bovine trypsin. Regions of functional importance are more strongly conserved. These include the active site residues asp102, his57, ser195, and the residue asp189 which is reputed to bind the basic residue at the substrate cleavage site. The activation peptide is not homologous to that of most vertebrate trypsins, suggesting a modified activation mechanism. The sequence further strengthens the hypothesis that the chymotrypsin cleavage specificity developed separately in the vertebrates and invertebrates.
95 citations
Book•
01 Jan 1985
TL;DR: This authoritative account of the developmental biology of genome multiplication, the reproduction of the genetic material that results in polyploid and polytene cells, is based on many years' study by its authors and is particularly valuable in that it includes scientific results previously available only in Russian.
Abstract: This authoritative account of the developmental biology of genome multiplication, the reproduction of the genetic material that results in polyploid and polytene cells, is based on many years' study by its authors. Polyploid and polytene cells regularly occur in a wide range of organisms, including mammals, invertebrates, plants and protozoa. The cells also have a particular significance for the function of the tissues and organs of which they are an integral part. The first part of the book details the origin of polyploidy and polyteny in the normal development of many tissue systems. In the second part the various modes of genome multiplication, its control, and its biological significance are discussed. The book is fully referenced citing literature published in many languages, and is particularly valuable in that it includes scientific results previously available only in Russian.
93 citations
TL;DR: Three gene-sized molecules cloned intact from macronuclear DNA served as hybridization probes to study excision of these molecules from chromosomes and their processing during macron nuclear development in the hypotrich Euplotes crassus.
75 citations
TL;DR: It is concluded that DNA replication in polytene cells is controlled by modifications of the mechanism operating in dividing cells, where control is sequence autonomous, and not by a “doubling of DNA” mechanism.
Abstract: In dividing cells, each sequence replicates exactly once in each S-phase, but in cells with polytene chromosomes, some sequences may replicate more than once or fail to replicate during S-phase. Because of this differential replication, the control of replication in polytene cells must have some unusual features. Dennhofer (1982a) has recently concluded that the total DNA content of the polytene cells of Drosophila salivary glands exactly doubles in each S-phase. This observation, along with previous studies demonstrating satellite underreplication in salivary gland cells, led us to consider the hypothesis that there is a "doubling of DNA" mechanism for the control of DNA replication in polytene cells. With this mechanism, a doubling of DNA content, rather than the replication of each sequence, would signal the end of a cycle of DNA replication. To test this hypothesis, we have reinvestigated the replication of several sequences (satellite, ribosomal, histone and telomere) in salivary gland cells using quantitative in situ hybridization. We find that underreplication of some sequences does occur. In addition we have repeated Dennhofer's cytophotometric and labeling studies. In contrast to Dennhofer, we find that the total DNA contents of nonreplicating nuclei do reflect this partial replication, in accord with Rudkin's (1969) result. We conclude that DNA replication in polytene cells is controlled by modifications of the mechanism operating in dividing cells, where control is sequence autonomous, and not by a "doubling of DNA" mechanism. In situ hybridization to unbroken salivary gland nuclei reveals the distribution of specific sequences. As expected, satellite, histone and 5S sequences are usually in a single cluster.(ABSTRACT TRUNCATED AT 250 WORDS)
70 citations
TL;DR: This work has developed techniques for synchronous mating of large populations of the hypotrichous ciliate Euplotes crassus and isolated DNA at various times of macronuclear development, finding that the average molecular weight of the DNA decreases at the time of chromosome transectioning.
Abstract: After conjugation in hypotrichous ciliates, a new macronucleus is produced from a copy of the micronucleus. This transformation involves large-scale reorganization of DNA, with conversion of the chromosomal micronuclear genome into short, gene-sized DNA molecules in the macronucleus. To study directly the changes that occur during this process, we have developed techniques for synchronous mating of large populations of the hypotrichous ciliate Euplotes crassus. Electron microscope studies show that the micronuclear chromosomes are polytenized during the first 20 h of macronuclear development. The polytene chromosomes lack the band-interband organization observed in other hypotrichs and in the Diptera. Polytenization is followed by transectioning of the chromosomes. We isolated DNA at various times of macronuclear development and found that the average molecular weight of the DNA decreases at the time of chromosome transectioning. In addition, we have shown that a small size group of macronuclear DNA molecules (450-550 base pairs) is excised from the chromosomal DNA approximately 10 h later in macronuclear development.
60 citations
TL;DR: Two largely independent studies of chromosomes from natural populations of anopheles maculatus provide evidence for several genetic species within the taxon, including two allopatric populations and heterochromatic variation occurs in both X and Y chromosomes.
Abstract: : Two largely independent studies of chromosomes from natural populations of anopheles maculatus provide evidence for several genetic species within the taxon. (1) Polytene chromosome variation shows four different rearrangements of arm 2 and three rearrangements of the X chromosome. There is strong evidence for three species. Two allopatric populations represent either dramatic geographic variation for two independent inversion systems within one of the genetic species, or represent two additional species. Their species status remains unresolved by this work. (2) Heterochromatic variation occurs in both X and Y chromosomes as revealed by Giemsa-banding of mitotic chromosomes from larval brains. The distribution and association of these various sex chromosomes give further evidence of a species complex. A preliminary correlation of these two kinds of chromosomal variation is given.
54 citations
TL;DR: The polytene chromosomes of larvae from samples of Eusimulium vernum, E. bicorne, and an undescribed species were examined and five of the total of six chromosome arms are involved in sex determination in the various members of this complex.
Abstract: The polytene chromosomes of larvae from samples of Eusimulium vernum, E. bicorne, and an undescribed species (designated here as Eusimulium "Yukon") were examined. Twelve cytotypes within E. vernum were distinguished, of which at least eight appear to be good biological species. These cytotypes, together with the two allied morphospecies, were related in a cytophylogeny. An ecological segregation between some of the siblings was observed. One cytotype apparently utilizes two (possibly three) separate chromosome (arms) in sex determination. Five of the total of six chromosome arms are involved in sex determination in the various members of this complex. The genetics of sex determination and the mechanisms of sex-locus shift are discussed in the context of these findings.
51 citations
TL;DR: Northern analysis of a genomic clone, lambda LS1, homologous to DmcMM115, indicates that two vitelline membrane genes may be clustered at the 26A site, and cross‐hybridization data suggest that DMCMM99 and Dmc MM115 are members of a multi‐gene family which includes at least three members.
Abstract: cDNA clones for two Drosophila vitelline membrane genes have been identified on the basis of: (i) stage and tissue specificity of transcription and (ii) size and amino acid content of the translation product. Cross-hybridization data suggest that DmcMM99 and DmcMM115 are members of a multi-gene family which includes at least three members, all of which reside on the left arm of the second chromosome. DmcMM99 and DmcMM115 originate from polytene band positions 34C and 26A, respectively. A third, cross-hybridizing gene resides at position 32EF. Southern analysis of a genomic clone, lambda LS1, homologous to DmcMM115, indicates that two vitelline membrane genes may be clustered at the 26A site.
TL;DR: Mapping of 16 regions of polytene chromosomes in which 18 one-band puffs develop was carried out with the use of electron microscopy, showing that active chromosome regions are rather diverse in both the pattern of decondensation and expansion of the decondensed region.
Abstract: Mapping of 16 regions of polytene chromosomes in which 18 one-band puffs develop was carried out with the use of electron microscopy (EM). In most cases a uniform decondensation of the whole band was observed. However, there were examples in which only a part of the band was activated (three puffs) or its right and left parts decondensed simultaneously (three puffs). Splitting of the band into two parts with their further decondensation was also found (one puff). This suggests structural and functional complexity of the bands. On the basis of the data obtained here and those published earlier, a classification of 52 puffs by the number of bands participating in their formation is given. Four classes numbering 22, 21, 7, 2 puffs, developing from 1, 2, 3 and 4 bands, respectively, are revealed. The data show that active chromosome regions are rather diverse in both the pattern of decondensation and expansion of the decondensed region, thus providing evidence of the informational complexity of the majority of active regions.
TL;DR: It is suggested that the class B clone, lambda Dm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.
Abstract: A cloned α-amylase cDNA sequence from the mouse is homologous to a small set of DNA sequences from Drosophila melanogaster under appropriate conditions of hybridization. A number of recombinant lambda phage that carry homologous Drosophila genomic DNA sequences were isolated using the mouse clone as a hybridization probe. Putative amylase clones hybridized in situ to one or the other of two distinct sites in polytene chromosome 2R and were assigned to one of two classes, A and B. Clone λDm32, representing class A, hybridizes within chromosome section 53CD. Clone λDm65 of class B hybridizes within section 54A1-B1. Clone λDm65 is homologous to a 1450- to 1500-nucleotide RNA species, which is sufficiently long to code for α-amylase. No RNA homologous to λDm32 was detected. We suggest that the class B clone, λDm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.
TL;DR: A method of mapping genes or transcripts on polytene chromosomes by transmission electron microscopy is described and evidence shows that these sites are localized in some bands and puffs, but are also found in interbands.
Abstract: We describe a method of mapping genes or transcripts on polytene chromosomes by transmission electron microscopy. We present several applications which illustrate that, in favorable cases, the method has a resolution of ca. 10 kb, and that high resolution mapping of hybridization sites relative to bands and puffs can be achieved. We mapped sites of transcription for poly-(A) RNA and present evidence which shows that these sites are localized in some bands and puffs, but are also found in interbands.
TL;DR: It is concluded that the vast majority of polytene chromosome bands (genes) contain one or a few DNA sequences with potential for undergoing the B----Z transition and contain both alternating purine-pyrimidine G-C and A-C tracts or mixed sequences.
Abstract: Anti-Z-DNA polyclonal and monoclonal immunoglobulins raised against left-handed polynucleotides show various degrees of specificity for base sequence and substitution Class 1 IgGs recognize all Z-DNA with equal affinity; class 2 IgGs show a preference for d(G-C)n sequences and class 3 IgGs for d(G-C)n sequences with substitutions at the C5 position of the pyrimidine These antibodies served as probes for the localization of Z-DNA in polytene and metaphase chromosomes and in interphase chromatin by indirect immunofluorescence A quantitative assessment of the binding of anti-Z-DNA IgGs to polytene chromosomes of Chironomus and Drosophila was made by scanning microphotometry and by computer-assisted image analysis of double immunofluorescence and DNA-specific dye fluorescence images The three classes of antibodies bind to most of the bands in acid fixed polytene chromosomes of C thummi; however, preferential binding of one class of antibody over another can be observed in certain regions These differences can be quantitated by arithmetic division or subtraction of the normalized digital images If a class 2 antibody is first bound at saturating concentrations the binding of class 1 antibody is reduced throughout most bands by 40-50% However, the telomeres of the three large chromosomes bind greater than 10 times as much class 1 antibody as class 2 antibody, indicating that the Z-DNA tracts in these regions are comprised largely of alternating sequences containing the A X T basepair, eg, A-C High-resolution image analysis of class 1 and class 2 immunofluorescence patterns and the total DNA distribution from polytene chromosomes of D melanogaster show that the two antibody distributions are very similar in a large majority of the bands, but they often deviate from the mean DNA distribution profile Z-DNA sequences of both G-C and A-C type are detectable at all levels of ploidy from 2n to 2(13)n and in species as diverse as insects and man We conclude that the vast majority of polytene chromosome bands (genes) contain one or a few DNA sequences with potential for undergoing the B----Z transition and contain both alternating purine-pyrimidine G-C and A-C tracts or mixed sequences Highly heterochromatic bands and telomeres have more Z potential sequences than do other bands
TL;DR: In polytene chromosomes of Drosophila hydei and D. melanogaster, Z-DNA was identified in varying distribution after different conditions for fixation were used, and was seen in portions of certain ecdysone- inducible puffs in the induced but not in the resting state.
Abstract: In polytene chromosomes of Drosophila hydei and D. melanogaster, Z-DNA was identified in varying distribution after different conditions for fixation were used. When salivary glands were fixed and squashed in 50% acetic acid alone, Z-DNA was found in the less dense DNA regions, such as interbands, some puffs, and a few of the less dense bands. Prefixation that combined ethanol and acetic acid exposure led to prominent immunofluorescent staining of the bands, generally but not strictly correlating with the total DNA content. Separate exposure to ethanol and acetic acid did not cause this band to stain, but if residual ethanol was present after ethanol fixation, subsequent exposure to acid did cause it. Under the more selective acid fixation conditions, Z-DNA reactivity was seen in portions of certain ecdysone-inducible puffs in the induced but not in the resting state; in other inducible regions, the Z-DNA immunoreactivity was not changed on induction. Z-DNA was also identified in polytene chromosomes within isolated nuclei that had been frozen and fixed in ethanol without exposure to acid; this Z-DNA was present in regions of low DNA density.
TL;DR: New data on rearrangements of ovarian polytene chromosomes from mosquitoes are presented for the following species in the series Neocellia of Anopheles (Cellia): annularis, philippinensis, nivipes, splendidus, jamesii, and ramsayi to suggest relationships between the species.
Abstract: New data on rearrangements of ovarian polytene chromosomes from mosquitoes are presented for the following species in the series Neocellia of Anopheles (Cellia): annularis, philippinensis, nivipes,...
TL;DR: The data support previous suggestions indicating a subsantial contribution of transcriptional products from small puffs and interbands to the whole transcriptional system of polytene chromosomes.
Abstract: Sites of transcriptional activity in the whole set of Drosophila melanogaster polytene chromosomes have been localized by means of fluorescent antibodies against DNA:RNA hybrid molecules and compared with results on 3H-uridine incorporation obtained earlier. — The majority of large and small puffs with intensive 3H-uridine incorporation demonstrate bright fluorescence. Moreover, bright fluorescence is also observed for a large number of small puffs though the intensity of 3H-uridine incorporation is low. Some prominent puffs with high levels of 3H-uridine incorporation show weak fluorescence. Condensed bands, as a rule, do not show fluorescence. — The regions that look like interbands under the light microscope are not real interbands, but consist of minibands visible only in the electron microscope (EM). However, a region that has been previously studied by EM and proven to be a real interband between two thick dark bands (100B3-100B4-5) showed fluorescence. These data support previous suggestions indicating a subsantial contribution of transcriptional products from small puffs and interbands to the whole transcriptional system of polytene chromosomes.
TL;DR: Whole genome Southern analysis showed that these genes belong to a large family of closely related calliphorin genes which were found by in situ hybridization to polytene chromosomes of trichogen cells to be clustered in region 4a of chromosome 2 of Calliphora vicina.
Abstract: A library of Calliphora vicina genomic DNA was constructed in the lambdaEMBL3 vector and screened for recombinant phages containing chromosomal segments encoding calliphorin, the major larval serum protein (LSP) of Calliphora. A large series of recombinants hybridizing with in vitro labelled poly(A) RNA from Calliphora larval fat bodies and with specific probes derived from the LSP-1 genes of Drosophila melanogaster was isolated. Five of these phages, chosen at random, were shown by hybrid selection to retain calliphorin mRNA specifically. Eleven calliphorin mRNA-homologous regions were located on restriction maps of these phages by hybridization with 5' end-labelled poly(A) RNA from Calliphora larval fat bodies. Each phage contains at least two calliphorin genes arranged in direct repeat orientation and seperated by 3.5-5 kb intergenic regions. The genes display similar but not identical restriction patterns. Filter hybridization and heteroduplex analysis indicate that they share a detectable homology with the LSP-1beta gene of D. melanogaster. Whole genome Southern analysis showed that these genes belong to a large family of closely related calliphorin genes which were found by in situ hybridization to polytene chromosomes of trichogen cells to be clustered in region 4a of chromosome 2 of Calliphora vicina.
TL;DR: Analysis of the band shape and of the fibrillar pattern in the interbands emphasizes that the polytene chromosome assumes a ribbonlike structure from which the more complex three-dimensional structure of the polyTene chromosome at higher levels of polyteny develops.
Abstract: Whole-mounted polytene chromosomes were isolated from nuclei by microdissection in 60% acetic acid and analyzed by electron microscopy. Elementary chromosome fibers in the interchromomeric regions and individual chromomeres can be distinguished in polytene chromosomes at low levels of polyteny (26–27 chromatids). Elementary fibers in the interbands are oriented parallel to the axis of the polytene chromosome. Their number roughly corresponds to the expected level of polyteny. These fibers have an irregular beaded structure, 100–300 A in diameter, and there is no apparent lateral association between them in the interchromomeric regions. Most bands, in contrast, form continuous structures crossing the entire width of the chromosome. Polytene chromosomes isolated in 2% or 10% acetic acid can be reversibly dispersed in a solution for chromatin spreading. The spread chromosomes consist of long uniform deoxyribonucleoprotein (DNP) fibers with a nucleosome structure. This supports the notion that continuous DNA molecules extend through the entire length of a polytene chromosome and that the nucleosome structure exists both in bands and interbands. Analysis of the band shape and of the fibrillar pattern in the interbands emphasizes that the polytene chromosome assumes a ribbonlike structure from which the more complex three-dimensional structure of the polytene chromosome at higher levels of polyteny develops.
TL;DR: Callus cultures of Nicotiana glauca, N. langsdorffii and of their tumor-forming hybrid plants contained a high frequency of cells with irregular chromosome numbers and chromosome aberrations, which suggest genome segregation in the cultures.
Abstract: Callus cultures of Nicotiana glauca, N. langsdorffii and of their tumor-forming hybrid plants contained a high frequency of cells with irregular chromosome numbers and chromosome aberrations (hypo-, hyper-, polyploid, aneuploid cells; bridges, polytene, broken, fragmented chromosomes, megachromosomes, etc.). Meristematic cells of shoot tips regenerated from the same cultures contained only regular chromosome numbers with normal chromosome structures. Variability in chromosome numbers is a consequence of abnormal mitoses. The data suggest genome segregation in the cultures. Cytological instability appears to be independent of genome segregation composition, genotype, tumorous condition, hormonal requirement and level of ploidy. The karyotype stability of the cultures is only dependent on the degree of organization of tissues and is regulated by factors involved in the control mechanisms of organizational processes.
TL;DR: Experiments indicate that the spontaneous excision of TE146, which results in the loss of the w+ and rst+ markers, can occur either as a single-step event or following a partial internal deletion, and despite their imprecise nature, all these spontaneous excisions restore a wild-type noc+ phenotype.
Abstract: The TE1 family of transposable elements (TEs) of Drosophila consists of unusually large transposons, cytologically visible in larval polytene chromosomes as one or more bands. They are composite elements, as their termini consist of foldback (FB) sequences which are themselves transposable. The location of FB elements at the termini of transposable elements suggests that these sequences have a direct role in the genetic instability of TEs. To investigate the structural and phenotypic consequence of TE excision, we have cloned genomic DNA required for the expression of the no-ocelli (noc) gene of Drosophila; this gene has been mutated by the insertion of TE146, a member of the TE1 family carrying six polytene chromosome bands including functional copies of the white (w+) and roughest (rst+) genes. As reported here, our experiments indicate that the spontaneous excision of TE146, which results in the loss of the w+ and rst+ markers, can occur either as a single-step event or following a partial internal deletion. In either case, the end product is an imprecise excision in which a residual portion of the element, varying in size from 3 to 10 kilobases (kb), is left at the insertion site. These residual sequences share homology with the FB family. Furthermore, despite their imprecise nature, all these spontaneous excisions restore a wild-type noc+ phenotype.
TL;DR: A library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana that carry sequences homologous to cDNA clones that hybridize in situ to the DNA puffs at the polytene chromosome regions C3 and C8 is constructed.
TL;DR: By applying some simple algorithms to these models, quantitative comparisons were made which revealed otherwise cryptic features common to many nuclei.
Abstract: SUMMARY
In Drosophila melanogater there are significant differences in the way the polytene chromosomes are arranged in different nuclei from the same salivary gland (Mathog et al., 1984). Visual inspection of the models of these nuclei was inadequate to delineate all of the features conserved in their structures. In order to bypass this limitation, models of these nuclei were constructed in a format compatible with computational manipulation (Mathog et al., 1985). By applying some simple algorithms to these models, quantitative comparisons were made which revealed otherwise cryptic features common to many nuclei. Given here are the details of several algorithms for describing and comparing the arrangement of the chromosomes within the nuclei.
TL;DR: The experiments on cultured pea roots (Pisum sativum cv. ‘Alaska’ and ‘Dwarf Telephone’) are summarized in Table 1 and polytene chromosomes were condensed into spherical structures, obviously a stage in their falling apart.
Abstract: The experiments on cultured pea roots (Pisum sativum cv ‘Alaska’ and ‘Dwarf Telephone’) are summarized in Table 1 Nuclear growth and mitosis occurred mainly in cultures in which the medium of Torrey and Shigemura was supplemented with 6 ppm 2,4-D and 1 ppm kinetin The greatest reaction was observed in 16-day cultures of cv ‘Alaska’ Nuclei had increased in size, and prophases diploid and polyploid, with normal chromosomes, diplochromosomes or larger bundles of chromatids were visible Metaphases which ranged from 2 n to an estimated 32 n had normal chromosomes with two chromatids Polytene chromosomes, in diploid, rarely in tetraploid number, occurred in numerous cortex cells They did not show banding, and their telomeres, spread into individual chromatids, were attached to the nuclear membrane In some cells the polytene chromosomes were condensed into spherical structures, obviously a stage in their falling apart; the last stage of this process is a polyploid metaphase
TL;DR: Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae, presenting numerous advantages over autoradiographic procedures.
Abstract: We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands, later in bands. Most chromosomal bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.
TL;DR: Mixtures of 17-base long oligonucleotides possibly encoding a hexapeptide of Drosophila melanogaster glucose-6-phosphate dehydrogenase were synthesized and used as probes for screening a genomic library of D. melanogasters constructed in Charon 4A vectors, and appeared versatile for isolating low-copy genes from genomic libraries.
Abstract: Mixtures of 17-base long oligonucleotides possibly encoding a hexapeptide of Drosophila melanogaster glucose-6-phosphate dehydrogenase were synthesized and used as probes for screening a genomic library of D melanogaster constructed in Charon 4A vectors A total of about 60, 000 plaques were initially screened, and after two successive plaque purification three clones carrying the identical 13-kb EcoRI fragment were isolated That these clones contain the G6PD coding sequence was demonstrated by in situ hybridization of the cloned DNA fragments to salivary gland polytene chromosomes and in vitro translation of the hybrid-selected mRNA As suggested by Torczynski et al (1984), this method using short synthetic probes appears versatile for isolating low-copy genes from genomic libraries
TL;DR: It has been established that 7-amino-actinomycin D induces a very weak fluorescence in GC-poor chromosome regions of the Drosophila lummei toromere structure.
Abstract: The cytochemical properties of a guanine-specific synthetic fluorescent analogue of actinomycin D, 7-amino-actinomycin D, have been studied in fixed and living preparations of L cells and polytene chromosomes of salivary glands ofChironomus thummi thummi andDrosophila lummei (Hackman).
TL;DR: The occurrence of a reverse transcriptase like enzyme in exconjugants of Stylonychia is described and a model is proposed according to which the gene-sized macronuclear DNA is cDNA synthesized from transcripts of the polytene chromosomes.
Abstract: During macronuclear development of hypotrichous ciliates over 90% of micronuclear sequences are eliminated and macronuclear DNA occurs in form of short gene-sized DNA molecules. In this paper the occurrence of a reverse transcriptase like enzyme in exconjugants ofStylonychia is described. A model is proposed according to which the gene-sized macronuclear DNA is cDNA synthesized from transcripts of the polytene chromosomes.
TL;DR: The results suggest that (1) dosage compensation involves changes in the template activity of the X chromosome and (2) the regulatory sequences involved in dosage compensation in Drosophila are located on the X chromosomes and are polygenic.
Abstract: The chromatin template activity of the polytene X chromosomal DNA was assayed by in situ transcription on the fixed polytene chromosomes using E. coli RNA polymerase holoenzyme and 3H-UTP as the monitoring substrate in various 1X2A, 2X2A and 3X2A larvae and 1X2A (+X fragments) segmental aneuploid larvae of Drosophila melanogaster. The segmental aneuploids contained duplications for the segments 15EF-20F, 11A-20F, 8C-20F and 3E-20F of the X chromosome. Results revealed that a double dose of active loci located in the X chromosome regions 15EF-20F, 11A-20F, and 8C-20F in aneuploids synthesized nearly 40%–70% more RNA than the normal single dose of this region in the wild-type males. The activity per gene dose for the two segments in the aneuploids was also significantly higher than in their male counterpart except for the duplication dp (3E-20F), where the duplicated piece extended from the centromeric heterochromatin to include 85% of the euchromatic portion of the X chromosome. In the case of dp (3E-20F), the X chromosome was transcribed at the lower, “female” level. It may also be noted that some regions of the X chromosome when present in extra copy, especially in dp (8C-20F) influenced the template activity of the X-linked genes inside or outside the duplicated segment. Metafemales (3X2A) have 50% higher template activity of the X chromosomes than their diploid sisters. In this study, metafemales behaved as females with duplication. These results suggest that (1) dosage compensation involves changes in the template activity of the X chromosome and (2) the regulatory sequences involved in dosage compensation in Drosophila are located on the X chromosome and are polygenic.