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J E Edström

Publications and source records attributed to J E Edström.

At least 55 records · Page 3Linked to original sources
PubMed1984

Cloning of a gene localized and expressed at the ecdysteroid regulated puff 74EF in salivary glands of Drosophila larvae.

The puffing cycle of salivary gland chromosomes of Drosophila larvae, which initiates the developmental path to pupariation, is induced by ecdysteroid hormone. Its action leads to prominent puffs at loci 2B5, 74EF and 75B. Fragments of the 74EF puff of the D. melanogaster 3L chromosome were microdissected from salivary gland squashes. EcoRI-digested DNA of these fragments was cloned into lambda phage. Clones were screened with puff stage-specific cDNA probes. Thirteen out of 650 clones hybridized preferentially with puff stage 4-specific cDNA. The prominent early puffs at 74EF and 75B are most active between puff stage 4 and 6. Therefore, one of the 13 lambda phages was chosen for further analysis. It was used to isolate 24 kb of Drosophila DNA from genomic libraries. The DNA hybridized in situ to locus 74F. The 74F DNA coded for a transcript, which was made in salivary glands, but not in fat body of third instar larvae. It accumulated in K(C) cells in response to ecdysteroid treatment. The polyadenylated transcript size was 2.7 kb as judged by Nothern blot analysis. The transcription start site of the 74F gene has been mapped. Sequences upstream of the transcription site contain several sequence elements common to other eucaryotic genes, including potential Z-DNA forming sequences. Also, there is sequence homology to upstream sequences, which have been involved in the regulation of transcription of the salivary gland glue protein 4 gene.

Journal Article↗
PubMed1984

Chromatin structure of a hyperactive secretory protein gene (in Balbiani ring 2) of Chironomus.

We examined the chromatin structure of a Balbiani ring (secretory protein gene) in the salivary glands of Chironomus larvae in its hyperactive state after stimulation with pilocarpine. For the inactive state of the gene an established tissue culture cell line, not expressing the gene, was used. Electron microscopy showed an RNA polymerase density of approximately 38/microns. Micrococcal nuclease digestion of purified nuclei followed by DNA transfer and hybridization revealed a smear with no recognizable discrete DNA fragments. Without pilocarpine stimulation a faint nucleosomal repeat was superimposed upon the smear, and in tissue culture cells a clear nucleosomal repeat was revealed. The restriction enzyme XbaI, which has a 6-bp recognition sequence, cut the gene in the hyperactive chromatin state, but not in its inactive conformation. The combined results are best explained by the absence of most of the nucleosomes in this hyperactive RNA polymerase II transcribed gene.

Animals↗
PubMed1984

Cloning of heat-shock locus 93D from Drosophila melanogaster.

Using the microcloning approach a number of recombinant lambda phages carrying DNA from the 93D region have been isolated. Screening genomic libraries, cloned in phage lambda or cosmid vectors, with this isolated DNA yielded a series of overlapping DNA fragments from the region 93D6-7 as shown by in situ hybridization to polytene chromosomes. In vitro 32P-labelled nuclear RNA prepared from heat-shocked third instar larvae hybridized specifically to one fragment within 85 kb of cloned DNA. The region which is specifically transcribed after heat shock could be defined to a cluster of internally-repetitive DNA and its neighbouring proximal sequences. Over a sequence of 10-12 kb in length the DNA is cut into repeat units of approximately 280 nucleotides by the restriction endonuclease TaqI. The TaqI repeat sequences are unique in the Drosophila genome.

Animals↗
PubMed1984

Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes.

Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.

Animals↗
PubMed1984

Balbiani ring induction in phosphate metabolism.

Balbiani rings (BR), giant puffs in Chironomus larval salivary glands, code for giant secretory proteins. As shown earlier, the normally dominant BR2 is turned off with its putative translation product during exposure of larvae to compounds that diminish the stores of P(i). A BR6 develops from a compact chromosome band, and a new giant protein appears in the secretion as the major component. We have determined the sequence of cloned DNA fragments representative for large parts of BR1 and BR2 (normally active) and the inducible BR6. There is an excess of positive charges and high contents of serine/threonine in the coded amino acid composition for the BR1 and BR2 sequences. The coded amino acid sequence for the BR6 clone shares homologies with the others but has an excess of negative charges and lacks serine/threonine. This suggested that the P(i) effects observed earlier could be related to differences in phosphorylation between the normal proteins and the BR6 product. This could be confirmed by measurements of phosphorylation, which occurs in the normal giant proteins mainly at seryl residues. P export with giant secretory protein is normally quantitatively important. Thus, BR6 activation should decrease P loss when P(i) pools are lowered because of inducer action.

Journal Article↗
PubMed1983

Molecular cloning of microdissected lampbrush loop DNA sequences of Drosophila hydei.

We microdissected a Y chromosomal lampbrush loop pair from primary spermatocyte nuclei of Drosophila hydei and cloned the DNA directly at the microscale. Four of the 12 recombinant DNA clones recovered display in situ hybridization to mitotic metaphase Y chromosomes, preferentially in the chromosomal region identified as the origin of the lampbrush loop pair. All clones, however, also hybridize to autosomal and X chromosomal loci in polytene chromosomes. Y chromosomal DNA sequences of D. hydei again prove to be members of different families of repeated sequences distributed throughout the genome. These microcloning experiments, which were carried out under very unfavourable experimental conditions (low DNA content of the lampbrush loops in the presence of large amounts of RNA) prove that almost any chromosomal structure detected by light microscopy is directly accessible to molecular cloning experiments by micromethods.

Journal Article↗
PubMed1983

Inhibition of Balbiani ring transcription following differential arrest of Balbiani ring-coded translation.

A differential inhibition of the synthesis of secretory proteins, mainly fractions formed in giant translation units, can be obtained in Chironomus salivary gland cells with low concentrations of the ribosome translocation inhibitor, cycloheximide with or without emetine. Both treatments also lead to puff regression and inhibition of transcription specific to the large Balbiani rings, BR1 and BR2, the loci for the giant secretory proteins. The amount of 75S BR RNA transcribed is also reduced in the cytoplasm and in the poly(A) RNA relative to other transcripts. The half-life of 75S RNA is, however, prolonged so that there is little if any decrease in the cytoplasmic content of 75S RNA. The effect on the Balbiani rings may be due to control emanating from the translational process.

Amino Acids↗
PubMed1983

Spread of Balbiani ring derived messenger RNA in Chironomus salivary gland cell cytoplasm.

We have investigated whether Balbian ring derived mRNA (75S RNA) of Chironomus salivary glands spreads to the different parts of the cytoplasm before or after attachment to the rough endoplasmic reticulum. Glands were fixed after in vivo administration of tritiated uridine and cytidine and the appearance of labelled RNA fractions in microdissected cytoplasm was measured. We chose central cytoplasm rich in rough endoplasmic reticulum (endoplasm) and peripheral cytoplasm with a low content of reticulum (basal zone). A peripheral spread of the mRNA on reticulum membranes would have moved the labelled RNA outwards with time. We found, however, a redistribution in the opposite direction, suggesting that the mRNA spreads over the whole cytoplasm before it attaches to the reticulum membranes. A similar redistribution occurs for the ribosomal subunits.

Animals↗
PubMed1982

Electron microscopic visualization of a discrete class of giant translation units in salivary gland cells of Chironomus tentans.

Lysates were made of whole salivary glands from Chironomus tentans and electron microscopic spreads prepared according to Miller and Beatty (1969). The sedimented material consisted mainly of ribosomes, most of which were present in giant polysomes which showed gradients of material protruding laterally from the ribosomes, interpreted as nascent polypeptide chains. Each giant polysome contained one such gradient. On the basis of the presence of a small group of terminal ribosomes without nascent chains at the 3' end, 19 apparently complete polysomes were selected, representing a discrete class of polysomes containing between 66 and 92 ribosomes (79 +/- 7, m +/- s.d.). Arguments are presented that they constitute the translation units for giant secretory proteins coded by RNA from the large Balbiani rings, BR1 and BR2.

Journal Article↗
PubMed1982

Constant and variable parts in the Balbiani ring 2 repeat unit and the translation termination region.

The large Balbiani rings of the chironomids produce giant internally repeated transcripts that are translated into silk-like proteins used for protective tubes. We have cloned fragments of the Balbiani ring 2 (BR2) gene of Chironomus pallidivittatus, normally the most prominent BR, for sequencing and restriction analysis. The results indicate a basic, tandemly arranged repeat unit of 198 bp, consisting of an invariant region of 102 bp followed by a variable region of 96 bp, the latter containing short internal tandem repeats. In the coding strand of both regions, there is a tendency to maximize adenine ( 40%) and minimize cytosine + thymidine (32-33%). Stringency in codon usage and absence of preference for third position nucleotide substitutions between homologous sequences suggest selection at the nucleotide level to be important. Both regions code for peptides rich in basic amino acids, but show distinct differences in other coding properties. The invariant region probably codes for a crystalline domain, and the variable region for a proline-rich, amorphous domain. One of the clones includes the 3' end of the translated region. Surrounding and following two stop codons is a sequence of four short palindromes. Furthermore, the first stop codon is part of a sequence reminiscent of a "TATA box". The possibilities are discussed that this area of the gene might be a target for regulatory molecules controlling translation termination and/or the expression of an overlapping cistron.

Journal Article↗
PubMed1981

Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes.

Fragments from section 3 of the salivary gland X chromosome of D. melanogaster were dissected with a micromanipulator. The DNA was extracted, cut and ligated to a lambda vector in a volume of a few nanoliters in an oil chamber monitored through a microscope. From about 10 pg of DNA we obtained 80 recombinant clones, a sample of which were analysed and shown to contain Drosophila DNA which hybridises in situ to the region of section 3 of the X chromosome. With this technique we can isolate clones from any desired region as small as 200 kb from the euchromatic arms of polytene chromosomes.

Bacteriophage lambda↗
PubMed1980

Large sized nascent protein as dominating component during protein synthesis in Chironomus salivary glands.

The main secretory protein fractions from Chironomus tentans have been investigated with particular emphasis on the dominant fraction, component 1, here designated I (Grossbach, 1969). This polypeptide was suggested to be the translatory product of 75SS RNA from Balbiani ring 2 (BR2) because of its size and quantitative prominence. Its molecular weight was estimated by gel filtration in 8 M urea at 850,000 + 101,000 D. During short pulses with radioactive amino acids a large fraction of the label was found in a population of polypeptide chains suggestive of molecules continuously growing to the size of compoenet I. Populations of nascent large protein chains of similar size distribution were dominant in the polysomes and constituted the only population present in the largest polysomes, known to contain 75S RNA from BR2 (and BR1) as predominant or only component (Daneholt et al., 1977; Wieslander and Daneholt, 1977). These data indicate strongly that the large size of component I is not a result of posttranslational modifications. No sequence similarities, using limited proteolysis, were found between component I and component II, both of which have been considered to the BR2 products. There was, furthermore, no detectable immunological identity between component I and smaller secretory protein fractions. The data support Grossbach's and Daneholt's suggestion that component I is closely related to the primary translation product of 75S RNA from the large Balbiani rings.

Amino Acid Sequence↗
PubMed1980

Sequences translated by Balbiani ring 75S RNA in vitro are present in giant secretory protein from Chironomus tentans.

The 75S RNA originating in the large Balbiani rings 1 and 2 (BRI and 2) was isolated and used for in vitro translation in the mRNA dependent reticulocyte lystate. Conditions (K+-concentration, temperature, time etc). were optimized for obtaining translation products of maximal size. Polypeptide chains up to about 500,000 D were obtained but no complete translation products. Trypic fingerprints were performed on the in vitro products as well as on the secretory protein components nos. I and II+III labelled with 35S-methionine. There was a large degree of correspondence between the fingerprint of the in vitro product and that of component I but less to that of component II+III. The results suggest that 75S RNA with an origin in the BR1 and BR2 codes for the giant secretory protein component I.

Amino Acid Sequence↗