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.     

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.     

In vitro transcription by isolated nuclei of Rhynchosciara americana salivary glands

A method for the isolation of polytene nuclei from salivary glands cells of the Diptera Rhynchosciara americana is described. The stage-specific morphological pattern of the chromosome is maintained during the isolation. The isolated nuclei show two distinct RNA polymerase activities, namely I and II, characterized on the basis of ionic requirements and -amanitin sensitivity. Studies of the product under the incubation conditions show that the system allows the synthesis of high-molecular weight RNA, beside a low molecular weight peak which may comprise pre-4S and 5S RNAs.-Autoradiographic studies carried out in the presence or absence of the toxin -amanitin showed that micronucleoli contain products of RNA polymerase type I activity (ribosomal RNA) and that the DNA puffs are engaged in -amanitin sensitive RNA synthesis and thus are sites of polymerase type II activity.     

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     

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'.     

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.     

Characterization of rapidly labelled RNA associated with DNA in Chlorella

Summary After pulse-labelling with ³²P-orthophosphate and fractionation of the nucleic acids from synchronously cultured cells of the green alga Chlorella pyrenoidosa on methylated serum albumin and kieselgur (MAK) the DNA contained a species of ³²P-RNA. About 3% of the total ³²P radioactivity incorporated in the cells' RNA were confined to this DNA-associated component. Its base ratio differed significantly from that of soluble and ribosomal RNA but varied only slightly during the life-cycle of the cells. About 4% of the DNA-associated ³²P-RNA resisted ribonuclease digestion suggesting a stable binding of RNA to DNA in the form of a complex. Gel filtration and sucrose gradient centrifugation of the nucleic acids isolated in the DNA region during previous MAK column chromatography resulted in a separation of most of the ³²P-RNA from the DNA. The remaining ribonuclease-resistent but alkali labile ³²P radioactivity bound to the latter was in the order of 4%. No evidence has been obtained so far that it represents rapidly synthesized RNA associated with DNA in stable and functional complex.