Characterisation of histones in the salivary glands of Rhynchosciara americana larvae

The histones from the salivary glands of Rhynchosciara americana larvae were identified. The electrophoretic patterns of the proteins studied resemble that of calf thymus histones, including the H₁ histone, which in Rhynchosciara has a lower electrophoretic mobility in urea/polyacrylamide gels but shows a molecular weight identical to the corresponding histone of calf thymus, as judged by SDS-polyacrylamide gel electrophoresis.     

Ecdysteroid titers and changes in chromosomal activity in the salivary glands of Rhynchosciara americana

Titers of ecdysone and 20-OH ecdysone were measured separately in both hemolymph and salivary glands of metamorphosing Rhynchosciara larvae. Gland titers were consistently higher than hemolymph titers. Although 20-OH ecdysone was the most prominent form of the hormone, measurable quantities of ecdysone were also observed throughout development in both tissues. Changes in salivary gland replication and puffing activity could be correlated with changes in gland 20-OH ecdysone titers. This was true for both developmentally changing RNA puffs and DNA puffs, which occur during the prepupal period. The DNA puffs are tied to the final DNA replication cycle, and both this cycle and the period of amplification can be correlated with increases in gland 20-OH ecdysone content. Various aspects and possible interpretations of the above correlations are discussed.This work is dedicated to the memory of Prof. Hans D. Berendes     

Messenger-like RNA synthesis and DNA chromosomal puffs in the salivary glands of Rhynchosciara americana

RNA synthesis was studied mainly in the proximal sections of Rhynchosciara salivary glands in late fourth instar at two typical periods of development. These are characterized either by the absence or presence of the so-called “DNA puffs” in the salivary gland chromosomes. It was found that simultaneously with the appearance of the DNA puffs there is a great increase in the synthesis of all RNA species. The greatest increase was found to take place in the rate of synthesis of messenger-like RNA. Four main classes of messenger-like RNA were detected, having mobilities corresponding to 33, 23, 16, and 14 S RNA. There is a correlation between the abundance of the 16 S messenger-like RNA and the degree of opening of the B-2 DNA puff. This species might therefore be transcribed from this puff.     

Relationships between newly synthesized proteins and DNA puff patterns in salivary glands of Rhynchosciara americana

An asymmetrical pericentric inversion in the onion fly, Hylemya antiqua was studied. Somatic pairing was studied in young eggs from test-and sibcrossed inversion heterozygous females which gave four and seven distinguishable karyotypes respectively. From these seven, three are balanced: the normal type, the inversion heterozygote and homozygote, and four are unbalanced recombinant karyotypes descending from crossovers in the loop. In all types at all mitotic stages the centromeres are paired. The telomeres only show association during prophase but this decreases from mid to late prophase. Quantitative analysis of the four different cross-over products as produced by inversion heterozygous females showed the presence of nonrandom disjunction. A significant disparity was observed, viz. the normal chromosome was taken up preferentially into the functional gamete compared to the inverted chromosome. Dragging of long chromatids in the asymmetric dyad during M I-A I is a possible explanation of this feature.     

Specialized features of Rhynchosciara americana embryogenesis

Insect embryo development is a complex process which requires nuclear and cellular division, cell shape alteration, and cell movement. This process needs to be orchestrated in a specific spatial and temporal fashion. Different insect species, despite similarities, present distinct morphogenetic pathways. We used the dipteran R. americana as a comparative model for embryo morphogenesis studies, following embryo development with different histochemical and immunohistochemical procedures. Despite the phylogenetic proximity with D. melanogaster, R. americana presents a peculiar morphogenesis. We show that at the initial phases of development, from egg fertilization to blastoderm formation, R. americana is similar to Drosophila. The first cleavages are nuclear and cellularization only begins after nuclei spread throughout the egg’s cortex. However after this stage a series of cell movements establishes a short compact germ band anlage, which gastrulates in a pattern quite different from Drosophila. After gastrulation the germ band elongates anterior–posteriorly and segmentation occurs simultaneously along the embryo. Embryo development from egg fertilization to larva hatching takes about 12 days. Our results show that R. americana presents a different morphogenetic pathway which does not fit in the current short, intermediate or long germ band classification.     

Ion and water transport by isolated cockroach salivary glands

Summary When the isolated salivary glands of the cockroachNauphoeta cinerea Olivier are stimulated by dopamine, the putative neurotransmitter, they secrete a fluid containing (mm): Na, 121; K, 47; Cl, 143. Stimulation of glands by 5-hydroxytryptamine or the neurotransmitter evokes a secretion identical in Na composition to this. Dopamine-evoked secretion is abolished in the absence of extracellular Na. The relationship between the rates of fluid secretion and Na transport is linear. However, at very low rates of secretion the Na concentration falls. Calcium, K and Cl ions can be removed from the bathing solution without abolishing fluid secretion. Our evidence suggests that (i) the primary secretion is formed by active transport of Na in the acini, and (ii) the ionic composition of this secretion is modified by re-absorption of Na and an independent secretion of K in the ducts.     

In vitro transcription of calliphorin genes in isolated nuclei of fat body from larvae of Calliphora vicina

Nuclei from fat body of different developmental stages of Calliphora vicina were isolated. They appear to be polyploid and show polytene chromosomes. The isolated nuclei were incubated with [³²P]GTP and the RNA transcribed in vitro was hybridized with a DNA fragment encoding a polypeptide subunit of calliphorin. The isolated nuclei transcribe the calliphorin-mRNA correctly and with the same stage specificity as observed in vivo.     

In vitro fatty acid acylation of mucus glycoprotein from sublingual salivary glands

The enzyme activity which catalyzes the transfer of palmitic acid from palmitoyl-coenzyme A to sublingual gland mucus glycoprotein has been demonstrated in the detergent extracts of the microsomal fraction of rat sublingual and parotid salivary glands. The acyltransferase activity of this fraction was similar in both types of glands. Further subcellular fractionation performed on sublingual glands revealed that the enzyme is associated with the Golgi-rich membrane fraction. Optimum enzymatic activity for fatty acylation of mucus glycoprotein was obtained using 0.5% Triton X-100, 2 mM dithiothreitol, 25 mM NaF, and 10 mM MgCl2 at a pH of 7.4. Higher concentrations of NaF, MgCl2 and dithiothreitol, however, were inhibitory. The apparent Km of the sublingual glands microsomal enzyme for mucus glycoprotein was 0.55 mg/ml and for palmitoyl-CoA, 3.5 X 10(-5) M. A 15% decrease in the acyltransferase activity was obtained with the reduced and alkylated mucus glycoprotein and it showed no activity towards the proteolytically degraded glycoprotein. The 14C-labeled product of the enzyme reaction gave in CsCl density gradient a band at the density of 1.49 in which the 14C label coincided with the glycoprotein. The 14C label in this glycoprotein was susceptible to deacylation with hydroxylamine, and the released labeled material was identified as palmitate.     

Histone synthesis in Rhynchosciara salivary glands. I. Site and timing of synthesis.

The site of histone synthesis was studied in polytene cells of the salivary glands of the Rhynchosciara americana (Diptera). It was found that, as is the case in non-polytene systems, these proteins are synthesized in the cytoplasm in a class of light polysomes which contain 3-4 ribosomes. This class of polyribosomes is most active at about 5 days before pupation when the nuclei are most active in DNA synthesis and the chromosomes of the gland show many open 'DNA puffs'.     

Dye-coupling between cells of the salivary glands in the cockroach Periplaneta americana

Cockroaches have acinar salivary glands. The acini consist of peripheral cells specialized for electrolyte and water transport and central cells contributing proteinaceous components to the saliva. Salivary duct cells probably modify the primary saliva. The acinar cells in Nauphoeta cinerea had been shown to be electrically coupled and dye-coupled. Since intercellular communication via gap junctions between acinar cells is difficult to reconcile with previous findings that dopamine and serotonin selectively stimulate the secretion of either protein-free or protein-rich saliva in Periplaneta americana, we investigated whether dye-coupling occurs between both acinar cell types and between duct cells. We iontophoretically loaded Lucifer yellow into impaled cells and tested for dye-coupling by confocal laser scanning microscopy. We found that: (1) peripheral and central cells within an acinar lobulus of the gland in P. americana are dye-coupled; and (2) salivary duct cells are dye-coupled.     

Embryonic expression of the engrailed homologue of Rhynchosciara americana

The segment polarity gene engrailed is involved in the determination of segment posterior identity in Drosophila. engrailed has been largely used for comparative developmental studies due to its evolutionary conservation from nematodes to humans. By in situ hybridization of an engrailed cDNA probe from Drosophila to polytene chromosomes of fourth instar larvae of Rhynchosciara americana we have shown that engrailed-like sequences must be localized in band 6 of chromosome A in this species. The pattern of engrailed protein expression during R. americana embryo development is diffuse at first evolving into a nuclear striped pattern after quite a length of time. In addition, our results suggest a possible developmentally regulated molecular modification of engrailed protein in R. americana embryos.