Looking at polytene chromosomes.
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Animals↗
Biomedical subjects
M L Pardue
Publications and source records attributed to M L Pardue.
The nucleus-limited Hsr-omega-n transcript is a polyadenylated RNA with a regulated intranuclear turnover.
The Drosophila Hsr-omega puff, one of the largest heat shock puffs, reveals a very unusual gene, identified by heat shock but constitutively active in nearly all cell types. Surprisingly, Hsr-omega yields two transcription end-products with very different roles. The larger, omega-n, is a nuclear RNA with characteristics suggesting a new class of nuclear RNAs. Although it neither leaves the nucleus nor undergoes processing, omega-n RNA is polyadenylated, showing that polyadenylation is not limited to cytoplasmic RNA, but possibly has a function in the nucleus. The amount of omega-n within the nucleus is specifically regulated by both transcription and turnover. Heat shock and several other agents cause rapid increases in omega-n. A rapid return to constitutive levels follows withdrawal of the agents. Degradation of omega-n is inhibited by actinomycin D, suggesting a novel intranuclear mechanism for RNA turnover. Within the nucleus, some omega-n RNA is concentrated at the transcription site; however, most is evenly distributed over the nucleus, showing no evidence of a concentration gradient which might be produced by simple diffusion from the site of transcription. Previous studies suggested that omega-n has a novel regulatory role in the nucleus. The actinomycin D-sensitive degradation system makes possible rapid changes in the amount of omega-n, allowing the putative regulatory activities to reflect cellular conditions at a given time. Omega-n differs from the best studied nuclear RNAs, snRNAs, in many ways. Omega-n demonstrates the existence of intranuclear mechanisms for RNA turnover and localization that may be used by a new class of nuclear RNAs.
Animals↗
Flightin, a novel myofibrillar protein of Drosophila stretch-activated muscles.
The indirect flight muscles of Drosophila are adapted for rapid oscillatory movements which depend on properties of the contractile apparatus itself. Flight muscles are stretch activated and the frequency of contraction in these muscles is independent of the rate of nerve impulses. Little is known about the molecular basis of these adaptations. We now report a novel protein that is found only in flight muscles and has, therefore, been named flightin. Although we detect only one gene (in polytene region 76D) for flightin, this protein has several isoforms (relative gel mobilities, 27-30 kD; pIs, 4.6-6.0). These isoforms appear to be created by posttranslational modifications. A subset of these isoforms is absent in newly emerged adults but appears when the adult develops the ability to fly. In intact muscles flightin is associated with the A band of the sarcomere, where evidence suggests it interacts with the myosin filaments. Computer database searches do not reveal extensive similarity to any known protein. However, the NH2-terminal 12 residues show similarity to the NH2-terminal sequence of actin, a region that interacts with myosin. These features suggest a role for flightin in the regulation of contraction, possibly by modulating actin-myosin interaction.
Actins↗
The Y chromosome of Drosophila melanogaster contains a distinctive subclass of Het-A-related repeats.
The HeT-A element is a transposable element with an apparent role in the structure of the telomeres of Drosophila melanogaster chromosomes. HeT-A transposition is the earliest event detected in healing of broken ends; HeT-A is also found on telomeres of unbroken chromosomes. Sequences with homology to HeT-A are never detected in euchromatic regions; however, clusters of HeT-A-related sequences occur in nontelomeric regions of the heterochromatic Y chromosome. Analysis of two of these Y-associated clusters shows them to be significantly different in structure from telomeric HeT-A elements, although the regions of shared sequence have > 80% sequence identity in all cases. Telomeric HeT-A elements occur in chains, with the elements in the same orientation but variably truncated at their external ends and irregularly interspersed with unrelated sequences. In contrast, the nontelomeric Y elements are regular tandem repeats of parts of the HeT-A sequence joined to unrelated sequences which are not the same in the two clusters studied. The sequence structures suggest that the nontelomeric clusters on the Y chromosome do not arise by the same transposition mechanism that forms the telomeric clusters; instead the clusters on the Y may arise by a mechanism that is used more generally in the evolution of Y chromosomes. Although the telomeric and nontelomeric clusters appear to be formed differently, both are enriched in parts of the HeT-A sequence which may be important in the structure of heterochromatin.
Animals↗
The heat shock response. Cells coping with transient stress.
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Animals↗
HeT-A, a transposable element specifically involved in "healing" broken chromosome ends in Drosophila melanogaster.
Eight terminally deleted Drosophila melanogaster chromosomes have now been found to be "healed." In each case, the healed chromosome end had acquired sequence from the HeT DNA family, a complex family of repeated sequences found only in telomeric and pericentric heterochromatin. The sequences were apparently added by transposition events involving no sequence homology. We now report that the sequences transposed in healing these chromosomes identify a novel transposable element, HeT-A, which makes up a subset of the HeT DNA family. Addition of HeT-A elements to broken chromosome ends appears to be polar. The proximal junction between each element and the broken chromosome end is an oligo(A) tract beginning 54 nucleotides downstream from a conserved AATAAA sequence on the strand running 5' to 3' from the chromosome end. The distal (telomeric) ends of HeT-A elements are variably truncated; however, we have not yet been able to determine the extreme distal sequence of a complete element. Our analysis covers approximately 2,600 nucleotides of the HeT-A element, beginning with the oligo(A) tract at one end. Sequence homology is strong (greater than 75% between all elements studied). Sequence may be conserved for DNA structure rather than for protein coding; even the most recently transposed HeT-A elements lack significant open reading frames in the region studied. Instead, the elements exhibit conserved short-range sequence repeats and periodic long-range variation in base composition. These conserved features suggest that HeT-A elements, although transposable elements, may have a structural role in telomere organization or maintenance.
Animals↗
Drosophila has a twitchin/titin-related gene that appears to encode projectin.
The sequences of twitchin and titin identify a superfamily of muscle proteins whose functions are not completely understood. In spite of their shared structural features, twitchin and titin appear to differ in function. Genetic and molecular evidence suggests that twitchin has a regulatory role in muscle contraction, whereas it has been proposed that titin has a structural function. We report here that Drosophila has a single-copy gene containing the two-motif amino acid sequence pattern that characterizes twitchin and titin. This gene appears to encode projectin, a muscle protein that is thought to play a structural role in asynchronous flight muscle but may have a role like that of twitchin in synchronous muscle. Thus Drosophila appears to be a case where the apparently diverged functions of twitchin and titin are encoded by a single gene.
Amino Acid Sequence↗
Dynamic instability of chromosomes and genomes.
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Animals↗
Structurally different Drosophila striated muscles utilize distinct variants of Z-band-associated proteins.
Monoclonal antibodies raised against four proteins from insect asynchronous flight muscle have been used to characterize the cross-reacting proteins in synchronous muscle of Drosophila melanogaster. Two proteins, alpha-actinin and Z(400/600), are found at the Z-band of every muscle examined. A larger variant of alpha-actinin is specific for the perforated Z-bands of supercontractile muscle. A third Z-band protein, Z(210), has a very limited distribution. It is found only in the asynchronous muscle and in the large cells of the jump muscle (tergal depressor of the trochanter). The absence of Z(210) from the anterior four small cells of the jump muscle demonstrates that cells within the same muscle do not have identical Z-band composition. The fourth protein, projectin, greater than 600 kDa polypeptide component of the connecting filaments in asynchronous muscle, is also detected in all synchronous muscles studied. Surprisingly, projectin is detected in the region of the thick filaments in synchronous muscles, rather than between the thick filaments and the Z-band, as in asynchronous muscles. Despite their different locations, the projectins of synchronous and asynchronous muscles are very similar, but not identical, as judged by SDS-PAGE and by peptide mapping. Projectin shows immunological cross-reactivity with twitchin, a nematode giant protein that is a component of the body wall A-band and shares similarities with vertebrate titin.
Actinin↗
Expression of heat-shock locus hsr-omega in nonstressed cells during development in Drosophila melanogaster.
The hsr-omega locus forms one of the largest Drosophila heat-shock puffs and produces three major transcripts. These three transcripts are also produced constitutively, at lower levels, in almost all tissues and developmental stages. The amounts of the transcripts in nonstressed cells are modulated during development. The hormone ecdysone leads to increased levels of hsr-omega transcripts in cultured cells, suggesting that changing ecdysone titers may play a role in the developmental changes of hsr-omega transcript levels. By in situ hybridization to RNA in tissue sections, we detect only two cell types that lack hsr-omega transcripts--the preblastoderm embryo and the primary spermatocyte. There are no maternal transcripts of hsr-omega in the embryo. Transcripts appear abruptly at the time that the zygotic genome becomes transcriptionally active, shortly before the formation of the cellular blastoderm. No constitutive hsr-omega transcripts are found in primary spermatocytes. The spermatocytes cannot respond to heat shock by transcribing either hsr-omega or hsp70 RNA. Constitutive hsr-omega transcription is resumed later in spermatogenesis and hsr-omega RNA is detected in differentiating spermatids. These spermatids are also capable of mounting a heat-shock response, as measured by increases in hsr-omega and hsp70 RNA.
Animals↗
Addition of telomere-associated HeT DNA sequences "heals" broken chromosome ends in Drosophila.
Stocks of D. melanogaster X chromosomes carrying terminal deletions (RT chromosomes) have been maintained for several years. Some of the chromosomes are slowly losing DNA from the broken ends (as expected if replication is incomplete) and show no telomere-associated DNA added to the receding ends. Two stocks carry chromosomes that have become "healed" and are no longer losing DNA. In both stocks the broken chromosome end has acquired a segment of HeT DNA, a family of complex repeats found only at telomeres and in pericentric heterochromatin. Although the HeT family is complex, the HeT sequence joined to the broken chromosome end is the same in both stocks. In contrast, the two chromosomes are broken in different places and have no detectable sequence similarity at the junction with the new DNA. Sequence analysis suggests that the new telomere sequences have been added by a specific mechanism that does not involve homologous recombination.
Animals↗
Heterochromatin: junk or collectors item?
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Animals↗
HeT DNA: a family of mosaic repeated sequences specific for heterochromatin in Drosophila melanogaster.
HeT DNA is a complex family of repeated DNA found only in pericentric and telomeric heterochromatin. In contrast to other DNA families that have been specifically associated with heterochromatin, HeT DNA is not principally a family of tandemly repeated elements. Much of the HeT DNA family appears to be a mosaic of several different classes of large sequence elements arranged in a scrambled array; however, some elements of the family can be found in tandem repeats. In spite of the variable order of the different elements in HeT DNA, the sequence homology between different members of each class of element is extremely high, suggesting that the members are evolving in a concerted fashion. Sequence analysis suggests that some elements in the HeT family may make up a novel family of heterochromatin-specific transposable elements and that the mosaic organization of the elements may be produced by retroposition and other mechanisms involved in the transposition of mobile elements. We suggest that such mechanisms may be a general feature for the maintenance of chromosome structure.
Animals↗
Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes.
He-T DNA is a complex set of repeated DNA sequences with sharply defined locations in the polytene chromosomes of Drosophila melanogaster. He-T sequences are found only in the chromocenter and in the terminal (telomere) band on each chromosome arm. Both of these regions appear to be heterochromatic and He-T sequences are never detected in the euchromatic arms of the chromosomes (Young et al. 1983). In the study reported here, in situ hybridization to metaphase chromosomes was used to study the association of He-T DNA with heterochromatic regions that are under-replicated in polytene chromosomes. Although the metaphase Y chromosome appears to be uniformly heterochromatic, He-T DNA hybridization is concentrated in the pericentric region of both normal and deleted Y chromosomes. He-T DNA hybridization is also concentrated in the pericentric regions of the autosomes. Much lower levels of He-T sequences were found in pericentric regions of normal X chromosomes; however compound X chromosomes, constructed by exchanges involving Y chromosomes, had large amounts of He-T DNA, presumably residual Y sequences. The apparent co-localization of He-T sequences with satellite DNAs in pericentric heterochromatin of metaphase chromosomes contrasts with the segregation of satellite DNA to alpha heterochromatin while He-T sequences hybridize to beta heterochromatin in polytene nuclei. This comparison suggests that satellite sequences do not exist as a single block within each chromosome but have interspersed regions of other sequences, including He-T DNA. If this is so, we assume that the satellite DNA blocks must associate during polytenization, leaving the interspersed sequences looped out to form beta heterochromatin. DNA from D. melanogaster has many restriction fragments with homology to He-T sequences. Some of these fragments are found only on the Y. Two of the repeated He-T family restriction fragments are found entirely on the short arm of the Y, predominantly in the pericentric region. Under conditions of moderate stringency, a subset of He-T DNA sequences cross-hybridizes with DNA from D. simulans and D. miranda. In each species, a large fraction of the cross-hybridizing sequences is on the Y chromosome.
Animals↗
Characterization of the gene for mp20: a Drosophila muscle protein that is not found in asynchronous oscillatory flight muscle.
A Drosophila melanogaster gene encoding a muscle specific protein was isolated by differential screening with RNA from primary cultures of myotubes. The gene encodes a 20-kD protein, muscle protein 20 (mp20), that is not detected in the asynchronous oscillatory flight muscles, but is found in most, if not all, other muscles (the synchronous muscles). The sequence of the protein, deduced from the DNA, contains two regions of 12 amino acids with significant similarity to high-affinity calcium-binding sites of other proteins. This protein is easily extracted from the contractile apparatus and thus does not seem to be a tightly bound structural component. The gene (located in polytene region 49F 9-13) is unique in the D. melanogaster genome and yields two transcripts, 1.0 and 0.9 kb long. The levels of the two transcripts are regulated differently during development, yet the coding regions of the two transcripts are identical.
Amino Acid Sequence↗
Multiple inducers of the Drosophila heat shock locus 93D (hsr omega): inducer-specific patterns of the three transcripts.
The Drosophila hsr omega locus produces one of the largest and most active heat shock puffs, yet it does not encode a heat shock protein. Instead, this locus produces a distinctive set of three transcripts, all from the same start site. The largest transcript, omega 1, is limited to the nucleus and appears to have a role there. A second nuclear transcript, omega 2, is produced by alternative termination and contains the sequence found in the 5' 20-25% of omega 1 (depending on the Drosophila species). The cytoplasmic transcript, omega 3, is produced by removal of a 700-bp intron from omega 2. All three hsr omega RNAs are produced constitutively and production is enhanced by heat shock. In addition to being a member of the set of heat shock puffs, the hsr omega puff is induced by agents that do not affect other heat shock loci, suggesting that hsr omega is more sensitive to environmental changes than other loci. We report here that agents that induce puffing of hsr omega loci in polytene nuclei also lead to an increase in hsr omega transcripts in diploid cells. We also show that the relative levels of omega 1 and omega 3 can be modulated independently by several agents. All drugs that inhibit translation, either initiation or elongation, stabilize the omega 3 transcript, which normally turns over within minutes in control cells. Drugs (such as benzamide and colchicine) that induce puffing of hsr omega, but not other heat shock loci, lead to large increases in omega 1. Although the constitutive level of omega 1 is relatively stable, the drug-induced excess is lost rapidly when the drug is withdrawn. The relative levels of hsr omega transcripts may reflect different states in cellular metabolism.
Animals↗
Unusual behavior of the cytoplasmic transcript of hsr omega: an abundant, stress-inducible RNA that is translated but yields no detectable protein product.
Although a major site of transcription in heat shock, the Drosophila hsr omega gene does not encode any known heat shock proteins. Instead, studies of the hsr omega transcripts suggest that the RNA molecules, rather than encoded proteins, are the active products of this gene. The cytoplasmic RNA, omega 3, is spliced and polyadenylated and yet has only very small open reading frames (ORFs), and these are poorly conserved in different Drosophila species. Surprisingly, the work reported here leads us to conclude that one of the tiny ORFs in this RNA is translated. This ORF, designated ORF-omega, is notable in being the only ORF that shows sequence conservation in the three Drosophila species examined. However, translation of this ORF does not lead to detectable accumulation of the protein product. We suggest that ORF-omega may be an example of an unusual type of translated ORF. The act of translation itself may be important rather than the generation of a functional protein product. This nonproductive translation may play a role in regulation of cellular activities.
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Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster.
Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-band components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies alpha:1-4 recognize a 90-100-kD protein which we identify as alpha-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate alpha-actinins. Monoclonal antibodies P:1-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A- and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (approximately 400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized alpha-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z- and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila alpha-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.
Actinin↗