Gene amplification in Rhynchosciara salivary gland chromosomes.

Abstract

Late in the fourth larval instar, several regions of the Rhynchosciara amercana salivary gland chromosomes undergo "DNA puffing. " We have constructed a library ofcloned cDNAs synthesized from poly(A)+RNA isolated from salivary glands'during the period ofdevelopment when the DNA puffs are active. From this library we have studied clones representative of three genes active during this period but not active at earlier developmental periods of the gland. One of these genes is not amplified during the developmental process and encodes a 0.6-kilobase RNA molecule. The other two genes are located within the DNA-puffsites C3 and C8and.encode 1.25-kilobase and 1.95-kldobase RNA molecules, respectively. We estimate -from the quantitation -of transfer hybridization experiments that each of these genes undergoes 16-fold amplification during DNA puffing.Gene amplification in somatic cells was first detected by morphological criteria in the larval salivary glands offlies ofthe family Sciaridae. Several regions of the Rhynchosciara americana polytene chromosomes were found to show a type ofpuffing in which, after puff regression, there was more DNA in the bands involved compared with neighboring bands as indicated by Feulgen staining (1). This was later confirmed. both by spectrophotometric measurements (2) and by autoradiographical studies on the incorporation of [3H]thymidine (3). Subsequently, similar observations-were made on the salivary chromosomes of larvae from the genus Sciara (4-6).The DNA puffs, which appear in late fourth instar in-Rhynchosciara salivary glands, are involved in the production of messenger RNAs (7,8). These encode several peptides of the communal cocoon, which are needed in large amounts over a short period of time (9, 10). Based on morphological and physiological criteria, the fourth instar of R. americana larvae has been divided into six periods (11). The first indication of DNA puff formation is the appearance of a fast-green staining band between two orcein (+) bands at the chromosomal sites ofthese puffs early in period 4 (ref. 12; unpublished data). Amplification and puffformation are subsequently maximal at different times for each site and are also dependent on the position of the cell within the gland (13, 14). The largest puffs are found in region 2 of the B chromosome and in regions 3 and 8 of the C chromosome. The B2 puffis formed preferentially in the first 50 cells of the gland in period 5, whereas C3 attains its largest size in the middle and distal section of the gland in period 6. The C8 puff is similar in all regions and is also maximal in period 6. As the larvae progress from period 3 to period 4; there is a dramatic change in the pattern of RNA and protein synthesis. This consists of an inhibition of rRNA synthesis (15) and the synthesis of new poly(A)+RNA species (7,8). This is accompanied by inhibition of the synthesis of certain peptides and the synthesis jtg of unfractionated RNA from the salivary glands of periods 3 and 5 larvae, was fractionated by electrophores...

Developmental control of the DNA replication and transcription programs

“…Moreover, gene expression in these tissues also likely occurs over a longer developmental window, and this could negate a need for amplification. Another example of developmental gene amplification occurs in the sciarid flies, in which genes encoding cocoon proteins are amplified in the larval salivary glands (Glover et al 1982;Wu et al 1993;Santelli et al 2004) In the case of the eggshell protein genes in the Drosophila follicle cells and the cocoon protein genes in the sciarid flies, it may be that when confronted with the developmental demand to express a limited set of genes at extremely high levels over a brief developmental window, increased copy number of a subset of genes above the overall ploidy of the genome has been selected.…”

Functional and bioinformatics analyses reveal conservation of cis‐regulatory elements between sciaridae and drosophilidae

“…These genomic regions constitute sites of developmentally regulated gene amplification and transcription (Basso et al, 2002;Foulk et al, 2006;Gerbi et al, 1993;Lara et al, 1991;Stocker et al, 1996). The formation of DNA puffs is induced by ecdysone and is concomitant with the transcription of the genes which present the highest levels of amplification within the amplicons (Fontes et al, 1992;Gerbi et al, 1993;Glover et al, 1982;Paçó-Larson et al, 1992;Penalva et al, 1997;Santelli et al, 1991Santelli et al, , 2004Wu et al, 1993;Yokosawa et al, 1999).…”

Bradysia hygida (Diptera, Sciaridae) presents two eukaryotic Elongation Factor 1A gene homologues: partial characterization of the eukaryotic Elongation Factor 1A-F1 gene

“…Taken together, these data further support the existence of two paralogous copies of the eEF1A gene in the nematoceran Dipteran B. hygida. Sciarid DNA puffs have been previously characterized as genomic regions in which developmentally regulated gene amplification occurs (33)(34)(35)(36)(37)(38). The only DNA puff-forming regions found on chromosomes A and X constitute the A14 and X4 regions, respectively (6).…”