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J M Amabis

Publications and source records attributed to J M Amabis.

14 recordsLinked to original sources
PubMed1997

Antibodies against the D-domain of a Chironomus ecdysone receptor protein react with DNA puff sites in Trichosia pubescens.

An antiserum (called AScE/D) against the semiconserved D-domain of a Chironomus tentans ecdysone receptor protein (cEcR) gave indirect immunofluorescence signals at DNA puff sites in Trichosia pubescens. The signals varied in maximum intensity at different DNA puff sites. Control experiments using the secondary rhodamine-labeled anti-rabbit IgG alone, preimmune serum, affinity purified AScE/D (called pABcE/D) and AScE/D preabsorbed with expressing bacterial extract or highly purified bacterially expressed cEcR indicated that the signals obtained at these chromosomal sites were likely to be due to specific interaction between an endogenous sciarid EcR and antibodies against cEcR. This conclusion was supported by observation of signals at certain Ec-inducible primary RNA puff sites. AScE/D signals began to appear at DNA puff sites during L3, the stage when amplification initiates, but at most sites their mean intensity was low and not statistically significant. Sites with AScE/D signals of significant mean intensity at this stage already showed evidence of transcription. The number and strength of transcription signals increased during L4. Comparison of the developmental course of signals for AScE/D, DNA synthesis, RNA presence/synthesis, and puff size for several DNA puffs during late larval- prepupal development showed a closer relationship of AScE/D signals with the initiation of RNA synthesis than with the initiation of DNA synthesis. Therefore, although we cannot absolutely eliminate a direct involvement of EcR in the amplification process at some sites, this investigation gives stronger support for its direct involvement in transcription. Since AScE/D signals are observed at DNA puff sites from the time the latter begin amplification/transcription through their regression, it appears that Ec and EcR are necessary as a sustained stimulus at these regions.

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PubMed1996

Ku-related antigens are associated with transcriptionally active loci in Chironomus polytene chromosomes.

Antigens of Chironomus reactive with human sera containing anti-Ku antibodies and also with specific antibodies to each Ku subunit were characterized by immunoblot analysis. Three main antigen species were identified in nuclear-enriched extracts from salivary gland cells of Chironomus thummi, ranging in Mr from 55000 to 67000. The nuclear localization of Ku-related antigens in the dipteran Chironomus was studied by immunofluorescent labeling in polytene chromosomes of the salivary glands. Balbiani rings, loci highly active in transcription, were found to be strongly labeled by anti-Ku antibodies. Sugar-induced changes in the activity of the Balbiani ring genes were accompanied by the redistribution of Ku-related antigens as visualized by their absence in regressed Balbiani ring loci, and continued presence only in those that were transcriptionally active. A drastic change in the distribution of Ku-related antigens was also observed when C. thummi larvae underwent heat treatment as the immunofluorescent staining was restricted to previously described heat shock puffs. Anti-Ku sera reacted in addition with several chromosomal bands in which the presence of RNA polymerase II was also immunologically detected. The results show that Chironomus antigens reactive with anti-Ku antibodies are related to transcription in polytene chromosomes.

Alcohols↗
PubMed1991

DNA sequence amplification in sciarid flies: results and perspectives.

The discovery of DNA sequence amplification in sciarid flies and investigations into its control and biological significance are reviewed. Results thus far show that amplification of specific salivary gland polytene chromosome bands is a general phenomenon in sciarids. It is brought about as part of a final endoreplication cycle by the rising titer of ecdysterone that occurs as the larvae approach the prepupal period. Amplification and transcription of these bands is a late, multistep effect of this hormone. The DNA puffs which form in amplified regions produce mRNAs which are translated into polypeptides that appear to be involved in cocoon formation. Application of molecular cloning techniques to the study of DNA amplification has allowed precise quantitation of amplification for several DNA puffs and is yielding maps of their transcription units. These techniques will ultimately help to define the origins of DNA puff replication and contribute to an understanding of the mechanism and control of the amplification phenomenon in Sciaridae. Projections for future experimental approaches are presented.

Animals↗
PubMed1990

The presence of an antigen reactive with a human autoantibody in Trichosia pubescens (Diptera: Sciaridae) and its association with certain transcriptionally active regions of the genome.

The antigens in HeLa and Trichosia pubescens cells, recognized by sera from patients with rheumatic diseases containing anti-Ku antibodies, were compared by means of immunoprecipitation of labeled cell extracts. The autoantibodies present in the tested sera precipitate at least two polypeptides of approximately Mr = 70,000 and Mr = 80,000 in HeLa cell extracts and a polypeptide of approximately Mr = 72,000 in Trichosia salivary gland cell extracts. The distribution of the insect antigen in chromatin was studied in salivary gland polytene chromosomes by indirect immunofluorescent staining with sera from two different patients. Both sera react with certain transcriptionally active chromosomal sites. The presence of the antigen in polytene chromosomes is strictly dependent on transcription, as no reaction is observed in the same sites before or after gene activation. Other sites, such as the nucleolar organizing region, are very active in transcription but never reacted with the anti-Ku positive sera. These results show that the insect antigen is associated with transcription-related processes of a subset of the chromosomal loci of T. pubescens. The anti-Ku positive sera react with a highly conserved antigen, which may serve a very important and similar role in the cellular metabolism of both insect and mammalian cells.

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PubMed1984

Effects of ecdysterone in polytene chromosomes of Trichosia pubescens.

Injection of ecdysterone into mid-fourth instar larvae of Trichosia pubescens brought about drastic changes in the puffing pattern of the chromosomes of the salivary gland. The puff sequence induced by the hormone is quite similar to that which normally occurs at the end of larval life. Three classes of puffs were observed: the early induced, the late induced, and those repressed by the hormone. Among the late-induced puffs, several were DNA puffs. The protein synthesis inhibition prevents the induction of all late puffs and the process of gene amplification but does not affect the induction of the early puffs.

Animals↗
PubMed1984

Hormonal control of gene amplification and transcription in the salivary gland chromosomes of Trichosia pubescens.

Trichosia pubescens larvae were ligated at different prepupal developmental stages in order to investigate whether ecdysterone acts just as a trigger or whether its presence in the hemolymph is constantly necessary for the induction of the late puffs, in particular the DNA puffs. The larvae were ligated in the middle of the body being sure that the salivary glands were divided in two halves. Some of the ligated larvae had the anterior portion of the body dissected immediately in order to determine the puff pattern at the time of ligature. These patterns were compared with those from the posterior half which were dissected at different times after ligature. The results obtained in these experiments can be summarized as follow: (1) the absence of ecdysterone source prevents the induction of late puffs even when ligature is carried out after the induction of the early ecdysterone-responsive puffs; (2) the lack of an ecdysterone source blocks the process of gene amplification when the ligature is carried out at the beginning of this process (L3 stage); (3) the gene amplification process would not seem to be blocked but DNA puffs do not expand if the larvae are ligated some hours later (early L4 stage); (4) both gene amplification and DNA puffs expansion occur normally when larvae are ligated at a more advanced stage (at late L4 stage or thereafter). These results show that the presence of ecdysterone source is necessary for the induction of the late puffs up to a critical period at L4 stage. They also show that gene amplification and transcription of amplified sequences at the DNA puffs are two independently controlled phenomena.

Animals↗
PubMed1982

Immunofluorescent characterization of DNA . RNA hybrids on polytene chromosomes of Trichosia pubescens (Diptera, sciaridae).

We have studied the distribution of DNA X RNA hybrids on polytene chromosomes with the aid of a goat antibody against DNA X RNA hybrids using the immunofluorescence technique. Fixed polytene chromosomes of the sciarid Trichosia pubescens (Diptera) show distinct, stage-specific labelling patterns throughout larval development. Controls for the staining procedure - including preincubation with hybrid-specific endoribonuclease H - prove that DNA X RNA hybrids are present on fixed chromosomes. They are revealed only under mild fixation conditions which do not efficiently immobilize all chromosomal proteins, indicating that some proteins have to be removed to make the antigens accessible to antibody. Certain fixation conditions may also cause local denaturation of chromosomal DNA, and some hybrids may possibly form during specimen preparation. After incorporation of radioactive uridine, a combination of phase contrast, fluorescent, and autoradiographic images of one and the same chromosomal preparation demonstrates that hybrid fluorescence is confined to transcriptionally active regions. Two puff classes can be distinguished. The first binds antibody and includes most RNA puffs and all DNA puffs so far studied; the second, comprising some RNA puffs, does not show bright fluorescence in spite of the fact that RNA synthesis is high as revealed by 3H-uridine incorporation. DNA X RNA hybrids are not found at DNA puff sites during the DNA amplification period; these sites contain detectable hybrids only when transcription is taking place. - Combination of the fluorescent technique with its excellent resolution and autoradiography should be helpful in studying detailed topological aspects of transcriptionally active chromosomal regions.

Animals↗
PubMed1979

Spermatogenesis in Trichosia pubescens (Diptera:Sciaridae).

This paper deals with the meiotic process in males of T. pubescens. The spermatocytes of this species are associated in clusters (rosettes) of some fifteen cells which are fairly synchronized during spermatogenesis. During meiosis, which is typically achiasmatic, 2 sets of chromosomes are eliminated into a narrow neck of cytoplasm (bud) which protrudes from the cell pole facing the centre of the rosette. The bud formation starts prior to the onset of the meiosis. The spindle that appears during the first meiotic division is unipolar with the fibres running from the pole at the end away from the bud up to the equatorial region of the cell. The chromosomes which move toward the spindle pole display a V-shape while the others, moving budward, do not show any consistent shape. We suggest that a special mechanism of chromosome motion is involved in the migration of these chromosomes. The second meiotic division is fairly similar to the ordinary type observed in other organisms except for the migration of both chromatids of one chromosome precociously toward the pole opposite the bud. At this division a typical bipolar spindle is present and all migrating chromosomes display a V-shape. The chromosomes eliminated during the first meiotic division clump together remaining in a pycnotic state at the distal portion of the bud until the end of spermatogenesis. The chromosomal set eliminated at the second meiotic division, as compared to the first, decondense simultaneously with the group located in the cell body forming a typical interphase nucleus. T. pubescens has 3 chromosomes limited to the germ-line cells. These chromosomes are typically heterochromatic, replicating their DNA out of phase with the S-chromosomes, probably at a later stage in interphase. Our results suggest that they are transcriptionally active in the interphase between the 2 meiotic divisions and just after the meiotic process.

Animals↗
PubMed1978

Experimental induction of gene activity in the salivary gland chromosomes of Trichosia pubescens (Diptera: Sciaridae).

During the course of experiments with larvae of Trichosia pubescens, we have unexpectedly found that diethyl ether or chloroform anesthesia induces a large puff in a specific band in the polytene chromosomes of the salivary glands. This puff develops a few minutes after the treatment, attaining its maximum size after 60-100 min, and regresses completely 200 min after its activation. Through autoradiography, an intense incorporation of RNA precursors into that puff was observed. A few other smaller puffs are also induced by the treatment. The treatment with diethyl ether or chloroform does not induce puffing in the polytene cells of malpighian tubules and of midgut.

Animals↗
PubMed1977

Cytological evidence of male heterogamety in sex determination of Telmatoscopus albipunctatus (Diptera: Psychodidae).

Telmatoscopus albipunctatus is polymorphic for several polytene chromosome bands. In examining the inheritance of a polymorphic heterochromatin-like band in chromosome IV we verified that it is inherited like a sex-linked factor. There are two types of chromosome IV in regard to this band: one bears a very thick heterochromatin-like band (H+), and the other bears a thinner corresponding band (H-). Three kinds of combinations are found in our stocks: H+H+, H+H- and H-H-. All three combinations can be found in females; however, in males, only the combinations H+H- and H-H- are found. Through specific crosses, it was concluded that the sex determining factor is located in chromosome IV in close vicinity to these bands.

Animals↗
PubMed1977

Puffing activity in the salivary gland chromosomes of Rhynchosciara under experimental conditions.

By using the techniques of ligation of the larvae (brain and endocrine glands extirpation) and salivary gland implantation, the hormonal dependence of the activity of certain puffs of Rhynchosciara was investigated. Our results have shown that the puffing behaviour--activation and deactivation--varies according to the developmental stage in which the larvae were ligated. When the larvae were ligated just before the drastic changes in the puffing pattern, which occur prior to pupation, these changes fail to occur. When the larvae were ligated after the onset of these changes we have observed: a) some of the puffs active at the time of the ligature regress promptly, earlier than their normal timing observed in controls; b) others remain active indefinitely and c) there are still some which regress accordingly to the normal timing. The puff B2 which behaves as those in b was double checked by means of implantation experiments. Salivary glands which had puff B2 at its maximum expansion were implanted into younger larvae and that puff also remained active in the body cavity of these larvae. Hypotheses to explain the results obtained are discussed.

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