F.J.S. Lara

Haemolymph amino acids and related compounds during cocoon production by the larvae of the fly, Rhynchosciara americana

Abstract A qualitative and quantitative analysis of free amino acids and related compounds in the haemolymph of Rhynchosciara americana was carried out for different periods of the fourth larval instar. Threonine, serine, proline, and glutamic acid make up 50 per cent of the total free amino acids in R. americana haemolymph just before the larvae start spinning the communal cocoon; after this the titre of most of the amino acids declines continuously. There are few peptides but these are present in high titres; they consist of two to three amino acid residues, of which the most important are histidine and aspartic acid. The fall in the haemolymph amino acid and peptide titres is insufficient to account for the silk protein which accumulates on the communal cocoon during the same period. The results are consistent with a silk protein origin from haemolymph proteins and haemolymph free amino acids. The origin and metabolic role of some haemolymph ninhydrin-positive compounds are discussed.

Relationships between newly synthesized proteins and dna puff patterns in salivary glands of rhynchosciara americana

An analysis of the polypeptides synthesized in the salivary glands of late fourth instar R. americana larvae was accomplished by the use of electrophoresis followed by fluorography of in vitro labelled proteins. It was possible to detect five polypeptides which are synthesized only when the giant DNA puffs occur. One of these polypeptides was tentatively assigned to DNA puff B2 and another to the DNA puff C3. This assignment was based on correlations of polypeptide labelling, puffing patterns and RNA synthesizing capacity of the puffs in different sections of the gland during development. The possible meanings and implications of these DNA puffs are discussed.

The giant DNA puffs of Rhynchosciara americana code for polypeptides of the salivary gland secretion

Abstract It was found that the polypeptides correlated with the appearance of DNA puffs B2 and C3 in the salivary gland of Rhynchosciara americana are present in the salivary secretion. This makes it very likely that these polypeptides are used in spinning the communal cocoon.

Molecular characterization of the C-3 DNA puff gene of Rhynchosciara americana.

We have mapped a region of about 33 kb which includes the transcription unit of the C-3 DNA puff gene of Rhynchosciara americana. The C-3 TU and a region extending approximately 800 bp upstream of the C-3 promoter were characterized. The TU is composed of three exons and produces a 1.1-kb mRNA whose level in salivary glands increases with the expansion of the C-3 puff. The C-3 messenger appears to undergo rapid deadenylation resulting in an RNA of about 0.95 kb which can still be observed in gland cells 15 h after the puff has regressed. The 1.1-kb mRNA codes for a 32.4-kDa, predominantly alpha-helical polypeptide with three conserved parallel coiled-coil stretches. The aa composition and structure of this polypeptide suggests that it is secreted and contributes to the formation of the cocoon in which the larvae pupate. The region upstream of the promoter contains several A-rich sequences with similarity to the ACS of yeast which might have a role in the initiation of replication/amplification.

RNA synthesis: a requirement for hormone-induced DNA amplification in Rhynchosciara americana

Injection of β-ecdysone into mid fourth instar larvae of Rhynchosciara americana induced within 23–28 hours after injection a rise in the percentage of 3H-thymidine (3H-TdR) incorporating nuclei in salivary gland region S1 from about 10–20% in the controls to 80–90% in the injected larvae. The 3H-TdR incorporating nuclei displayed a weak continuous labeling pattern or a band-labeling pattern with grains over the vast majority of the bands. The majority of nuclei with a band labeling pattern displayed DNA amplification at the DNA-puff regions. — Injection of actinomycin D at different times after ecdysone injection abolished the higher incorporation rate at the amplifying regions within 15 hours after the injection. However, the percentage of nuclei incorporating 3H-TdR and the frequency of the two labeling patterns remained essentially the same when RNA synthesis was inhibited. Only the over-all rate of 3H-TdR incorporation seemed to be reduced. — These data suggest that in the DNA puff regions the rate of DNA chain elongation is higher when amplification occurs than during a normal replication cycle. It, further, seems that the higher rate during amplification is dependent upon de novo RNA synthesis.

Molecular characterization of the B-2 DNA puff gene of Rhynchosciara americana

We have sequenced a 2.5-kb DNA fragment of the B-2 DNA puff from the sciarid Rhynchosciara americana and have defined its transcription unit. This puff is active during the formation of the communal cocoon, which is important for successful metamorphosis of this species and coincides with the final cycle of polytenization in its salivary glands. The B-2 polypeptide, together with the products of two other previously characterized DNA puffs, seems to be engaged in an interaction that results in a gradual modification and hardening of the cocoon structure. The B-2 messenger is temporally regulated in apparent coordination with the other puff products. The predicted polypeptide has characteristics similar to polypeptides from previously sequenced DNA puff genes, in particular those from the R. americana C-8 gene and the Bradysia hygida C-4 gene. The cloned sequence of the B-2 puff is differentially amplified in the three gland regions examined, achieving its highest amplification level of approximately fourfold (two extra cycles) in the anterior segment of the gland. The C-3 DNA puff sequence was also found to be differentially amplified in the different gland regions. Implications of the widespread presence of DNA amplification as a form of gene regulation in the Sciaridae are discussed.

Replication and transcription in the course of DNA amplification of the C3 and C8 DNA puffs of Rhynchosciara americana

The synchronous development of a sibling group of Rhynchosciara larvae enables us to follow the relationship between the local transcription and extrareplication of C3 and C8 DNA puffs. Although DNA amplification at these two loci takes place during the last cycle of DNA duplication in salivary gland chromosomes, a different timing of puff expression was observed for the two regions analysed. C3 puff transcription is a late event in relation to the C8 counterpart. We present evidence that this might be a consequence of the different firing of DNA amplification in both loci. No signs of DNA rearrangements were detected with probes that extend the previously analysed regions.

DNA replication during amplification of the C3 puff of Rhynchosciara americana initiates at multiple sites in a 6 kb region

Abstract.Two independent two-dimensional agarose gel electrophoresis methods have been used to map the origin of replication that directs amplification of the C3 DNA puff of Rhynchosciaraamericana. The results of neutral/neutral two-dimensional gel electrophoresis show that DNA replication initiates at multiple sites in a zone of at least 6 kb situated immediately upstream from the promoter of the main transcription unit of this puff. The complementary neutral/alkaline two-dimensional gel electrophoresis technique shows that, within the initiation zone, forks move in both directions. In contrast, unidirectional fork movement away from the initiation zone is observed at the ends of the region, implying that it is the only place in the amplified region of the C3 puff where initiations occur. Since the initiation zone coincides with the region that is most highly amplified, amplification of the C3 puff probably occurs by an onion skin-type mechanism.

Immunofluorescent characterization of DNA·RNA hybrids on polytene chromosomes of Trichosia pubescens (Diptera, Sciaridae)

We have studied the distribution of DNA·RNA hybrids on polytene chromosomes with the aid of a goat antibody against DNA· 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·RNA hybrids are present on fixed chromosomes. They are revealed only under mild fixation conditions which do not efficiently immobilise 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·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.

The transcript from a DNA puff of Rhynchosciara and its migration to the cytoplasm

Electrophoretic analysis of 3H-RNA obtained from the proximal sections of Rhynchosciara salivary glands at two distinct developmental periods, one characterized by the presence and the other by the absence of the giant B-2 DNA puff, revealed that the appearance of a 14S poly(A)+ RNA is correlated with the opening of this puff. That this RNA species is transcribed from this puff is indicated by the fact that it is found in RNA extracted from B-2 puffs obtained by microdissection. This confirmed by the specific hybridization of the 14S poly(A)+ RNA to the B-2 locus. Our data indicate that the polyadenylation process takes place at the chromosome level, and that the nuclear sap is not an important compartment in the transport of polyadenylated RNA from the chromosome to the cytoplasm. The kinetics of migration of the 14S species to the cytoplasm were studied; the data indicate that this process is very rapid and, in addition, that the 14S RNA is unstable.