Rapid and high resolution detection of in situ hybridisation to polytene chromosomes using fluorochrome-labeled RNA

Fluorochrome-labeled RNA allows the rapid detection of in situ hybrids without the need for long exposure times as in the autoradiographical hybridisation methods. Resolution is high because of the high resolving power of fluorescence microscopy. The application of a previously reported method for the hybrido-cytochemical detection of DNA sequences to polytene chromosomes of Drosophilia is described. — The specificity and sensitivity of the method are demonstrated by the hybridisation with polytene chromosomes of 1) rhodamine-labeled 5S RNA, to the 5S rRNA sites of D. melanogaster (56F) and D. hydei (23 B), 2) rhodamine-labeled RNA complementary to a plasmid containing histone genes, to the 39DE region of D. melanogaster, 3) rhodamine-labeled D. melanogaster tRNA species (Gly-3 and Arg-2), to their respective loci in D. melanogaster, 4) rhodamine-labeled RNA complementary to the insert of plasmid 232.1 containing part of a D. melanogaster heat shock gene from locus 87 C, to D. hydei heat shock locus 2-32A. In the latter instance it was possible to demonstrate the labeling of a double band which escaped unambiguous detection by autoradiography in the radioactive cytochemical hybridisation procedure because of the low topological resolution of autoradiograms. — The sensitivity of the fluorochrome-labeled RNA method is compared with the radioactive methods which use ³H- or ¹²⁵I-labeled RNAs. The factors governing the sensitivity and the number of bound fluorochrome molecules to be expected are discussed.Dedicated to Professor W. Beermann in honour of his 60th birthday     

Isolation and sequence analysis of sea urchin (Lytechinus pictus) histone H4 messenger RNA

Histone messenger RNAs isolated from early blastula stage Lytechinus pictus sea urchin embryos have been separated into discrete RNA bands on polyacrylamide gels. The most rapidly migrating of these molecules, the putative histone H4 mRNA, has been digested with T₁ ribonuclease to generate oligonucleotides for nucleotide sequence analysis. Many of these sequences are colinear with the highly conserved amino acid sequence of histone H4 protein as determined for both cows and peas.Histone H4 messenger RNA hybridizes in conditions of DNA excess to sea urchin DNA which is repeated approximately 470-fold. Despite this level of repetition the nucleotide sequence of the H4 messenger RNA reflects little evolutionary divergence within the H4 genes of L. pictus as judged by the stoichiometric yield of T₁ oligonucleotides and the hybridization and thermal stability of histone H4 mRNA-DNA hybrids.     

The organization of sea urchin histone genes

Sucrose gradient analysis of total sea urchin DNA cleaved with theEcoRI andHind III restriction endonucleases and identification of histone coding gene sequences by hybridization with histone mRNA have elucidated the basic organization of the histone gene repeat unit. These data, plus results obtained by electrophoretic analysis of purified endonuclease-cleaved sea urchin histone DNA and hybridization with cRNA transcribed from the eucaryotic segment of constructed plasmid chimeras cloned in E. coli, show that the several DNA sequences coding for individual histone proteins are intermingled in a 7 kilobase (kb) repeat unit. Cleavage of total sea urchin DNA withEcoRI produces 2.2 and 4.8 kb fragments which are homologous with the two cloned fragments, and which are contained in a 7 kbHind III fragment. Cleavage with both enzymes reveals that the 2.2 kbEcoRI fragment contains aHind III site 0.15–0.2 kb from an end. RNA · DNA hybridization between chimeric plasmid DNA and purified individual mRNAs isolated from sea urchin embryo polyribosomes has been used to assign coding sequences to either the 2.2 or 4.8 kb region of the histone DNA repeat unit. A map of the histone genes is proposed.     

Adjacent chromosomal regions can evolve at very different rates: Evolution of theDrosophila 68C glue gene cluster

Summary The 68C puff is a highly transcribed region of theDrosophila melanogaster salivary gland polytene chromosomes. Three different classes of messenger RNA originate in a 5000-bp region in the puff; each class is translated to one of the salivary gland glue proteins sgs-3, sgs-7, or sgs-8. These messenger RNA classes are coordinately controlled, with each RNA appearing in the third larval instar and disappearing at the time of puparium formation. Their disappearance is initiated by the action of the steroid hormone ecdysterone. In the work reported here, we studied evolution of this hormone-regulated gene cluster in themelanogaster species subgroup ofDrosophila. Genome blot hybridization experiments showed that five other species of this subgroup have DNA sequences that hybridize toD. melanogaster 68C sequences, and that these sequences are divided into a highly conserved region, which does not contain the glue genes, and an extraordinarily diverged region, which does. Molecular cloning of this DNA fromD. simulans, D. erecta, D. yakuba, andD. teissieri confirmed the division of the region into a slowly and a rapidly evolving protion, and also showed that the rapidly evolving region of each species codes for third instar larval salivary gland RNAs homologous to theD. melanogaster glue mRNAs. The highly conserved region is at least 13,000 bp long, and is not known to code for any RNAs.     

Expression of maternal and paternal histone genes during early cleavage stages of the echinoderm hybrid Strongylocentrotus purpuratus × Lytechinus pictus

Interspecific hybrids of the sea urchins Strongylocentrotus purpuratus (♀) and Lytechinus pictus (♂) were used to estimate the contributions of the maternal and paternal genomes to histone mRNA synthesis during early development. Radiolabeled histone mRNAs from the two sea urchin species were identified by hybridization to cloned histone genes from both S. purpuratus and L. pictus and shown to be electrophoretically distinguishable. The synthesis of maternal and paternal histone mRNA in these hybrid embryos is evident as early as the two-cell stage. By at least the 16-cell stage, both maternal and paternal histone mRNAs are associated with polysomes. The relative amounts of the maternal and paternal histone mRNAs synthesized by the zygote appear to be similar.     

Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

Histone Gene Multiplicity and Position Effect Variegation in DROSOPHILA MELANOGASTER

The histone genes of wild-type Drosophila melanogaster are reiterated 100–150 times per haploid genome and are located in the segment of chromosome 2 that corresponds to polytene bands 39D2-3 to E1-2. The influence of altered histone gene multiplicity on chromatin structure has been assayed by measuring modification of the gene inactivation associated with position effect variegation in genotypes bearing deletions of the 39D-E segment. The proportion of cells in which a variegating gene is active is increased in genotypes that are heterozygous for a deficiency that removes the histone gene complex. Deletions that remove segments adjacent to the histone gene complex have no effect on the expression of variegating genes. Suppression of position effect variegation associated with reduction of histone gene multiplicity applies to both X-linked and autosomal variegating genes. Position effects exerted by both autosomal and sex-chromosome heterochromatin were suppressible by deletions of the histone gene complex. The suppression was independent of the presence of the Y chromosome. A deficiency that deletes only the distal portion of the histone gene complex also has the ability to suppress position effect variegation. Duplication of the histone gene complex did not enhance position effect variegation. Deletion or duplication of the histone gene complex in the maternal genome had no effect on the extent of variegation in progeny whose histone gene multiplicity was normal. These results are discussed with respect to current knowledge of the organization of the histone gene complex and control of its expression.     

Heat-shock DNA homology in distantly related species of Drosophila

Polytene chromosomes of D. melanogaster and D. virilis were hybridized in situ with ¹²⁵I labeled mRNA isolated from polysomes of D. melanogaster tissue culture cells incubated at 37° C. ¹²⁵I mRNA hybridized preferentially with subdivisions 87A and 87Cl of the D. melanogaster 3R chromosome; grains were also observed at regions 93D, 95D and over the chromocenter. A considerable cross hybridization of this mRNA with D. virilis polytene chromosomes was observed. The 29C region of the D. virilis second chromosome was the main site of hybridization. Significant grain numbers also appeared in region 20F of the same chromosome. The two regions mentioned belong to heat shock loci in the latter species. Based on label intensity we conclude that region 29C of D. virilis contains DNA sequences retaining molecular homology with those at subdivisions 87A and 87Cl of D. melanogaster. SDS-polyacrylamide gel electrophoresis revealed similar distributions of heat shock proteins in the two species studied.     

Sequence analysis and evolution of sea urchin (Lytechinus pictus and Strongylocentrotus purpuratus) histone H4 messenger RNAs

The putative histone H4 (F2a₁) mRNA has been isolated from early blastula Strongylocentrotus purpuratus sea urchin embryos. Nucleotide sequences of oligonucleotides obtained by digestion of this RNA with T₁ ribonuclease have been obtained and many are found to be colinear with the amino acid sequence of histone H4 protein. The sequences obtained from the H4 mRNAs of S. pnrpuratus have been compared with those obtained from Lytechinus pictus (Grunstein & Schedl, 1976). The two mRNAs for this highly conserved protein have undergone considerable divergence of the sort that would be predicted from the degeneracy of the genetic code. 11.5% of the bases have undergone substitution at a rate calculated to be 3 × 10^?9 base changes · codon^?1 · year^?1.     

Evidences of two different sets of histone genes active during embryogenesis of the sea urchin Paracentrotus lividus.

Histone mRNAs at different stages of development were purified by hybridization with the cloned homologous histone genes. The electrophoretic patterns of oocytes, 2-4 blastomeres, 64 cells and morula histone mRNAs was found to be identical, whereas the electrophoretic pattern of mesenchyme blastula histone mRNA was markedly different. The cloned histone DNA of P.lividus was hybridized with the RNA of each stage. The Tm was 74 degrees C in all cases except for the mesenchyme histone mRNAs whose Tm was 59 degrees C, thus suggesting that at least two different clusters of histone genes are active in the course of the sea urchin development.     

Construction of chimeric plasmids containing histone H5 cDNA from hen erythrocyte. DNA sequence of a fragment derived from the 5' region of H5 mRNA.

We report that construction and characterization of chicken erythrocyte histone H5 cDNA recombinant plasmids. cDNA was synthesized from poly(A)+ polysomal RNA enriched in H5 mRNA and inserted into the PstI site of pBR322. Several clones containing H5 cDNA sequences were obtained and one of them (p541), expressing H5 antigenic determinants, was sequenced. The DNA insert of p541 contains 118 nucleotides from the 5' non-translated region of H5 mRNA and sequences coding for up to residue 46 of the N-terminus of the arginine (position 15) H5 variant. There is a strikingly high number of repeated sequences both in the leader and coding region; among these, the octanucleotide 5' GCG GCG GC 3' is found five times along the sequence. Although the H5 mRNA 5' leader is GC-rich (66%), there is an AT-rich region, about 16 nucleotides long, which shares strong homology with the leaders of sea urchin histone H1 mRNAs.     

Fluorescence patterns of heterochromatin in mitotic and polytene chromosomes in seven members of three sub-groups of the melanogaster species group of Drosophila

A comparative study of fluorescence patterns of heterochromatin in mitotic and polytene chromosomes of seven species belonging to 3 subgroups melanogaster sub-group: D. melanogaster and D. simulans; montium sub-group: D. kikkawai and D. jambulina; ananassae sub-group: D. ananassae, D. malerkotliana and D. bipectinata) of the melanogaster species group of Drosophila (Sophophora) has been made. Hoechst 33258 (H) fluorescence patterns of mitotic chromosomes reveal differences correlated to the taxonomic groupings of these species. The melanogaster sub-group species have H-bright regions on heterochromatin of all chromosomes; the montium subgroup species have H-bright regions mainly on the 4th and Y-chromosomes; in the ananassae sub-group, while D. ananassae chromosomes do not show any H-bright regions, D. malerkotliana and D. bipectinata have small H-bright segments only on their 4th chromosomes. The H-and quinacrine mustard (QM) fluorescence patterns of larval salivary gland polytene chromocentre in these species, however, do not show the same taxonomic correlation. While D. ananassae and D. kikkawai polytene nuclei lack any H-or QMbright region in the chromocentre, the remaining species have prominent H-and/or QM-bright region(s). In D. jambulina, the QM-bright regions are generally bigger than H-bright regions, while in D. malerkotliana and D. bipectinata the situation is reversed. Actinomycin D counterstaining prior to H-staining of polytene preparations of each species confirms that the H-bright region/s in the chromocentre are composed of A-T rich sequences. In vivo labelling of salivary gland polytene nuclei with 5-bromodeoxyuridine for 24 to 48 h and subsequent H-staining reveals that in all the species, the H-bright regions do not replicate in 3rd instar stage and presumably represent the non-replicating alpha heterochromatin. Significantly, in all the species (excepting D. kikkawai and D. ananassae), the size, location and the number of H-and/or QM-bright regions were seen to vary in different polytene nuclei in the same gland. It seems that the organization and the extent of under-replication of alpha heterochromatin varies in different polytene nuclei. Present studies also show that even closely related species differ in the content and organization of H-bright heterochromatin. The 81 F band at the base of 3 R in D. melanogaster, but not in D. simulans, appears to contain non-replicating H-bright sequences in addition to replicating chromatin.