The circadian mechanism appears remarkably conserved between Drosophila and mammals, with basic underlying negative and positive feedback loops, cycling gene products, and temporally regulated nuclear transport involving a few key proteins. One of these negative regulators is PERIOD, which in Drosophila shows very similar temporal and spatial regulation to TIMELESS. Surprisingly, we observe that in the housefly, Musca domestica, PER does not cycle in Western blots of head extracts, in contrast to the TIM protein. Furthermore, immunocytochemical (ICC) localization using enzymatic staining procedures reveals that PER is not localized to the nucleus of any neurons within the brain at any circadian time, as recently observed for several nondipteran insects. However, with confocal analysis, immunofluorescence reveals a very different picture and provides an initial comparison of PER/TIM-containing cells in Musca and Drosophila, which shows some significant differences, but many similarities. Thus, even in closely related Diptera, there is considerable evolutionary flexibility in the number and spatial organization of clock cells and, indeed, in the expression patterns of clock products in these cells, although the underlying framework is similar.
Photoperiodic signal stimulates induction of larval diapause in Chymomyza costata. Larvae of NPD strain (npd-mutants) do not respond to photoperiod. Our previous results indicated that the locus npd could code for the timeless gene and its product might represent a molecular link between circadian and photoperiodic clock systems. Here we present results of tim mRNA (real time-PCR) and TIM protein (immunohistochemistry) analyses in the larval brain. TIM protein was localized in 2 neurons of each brain hemisphere of the 4-d-old 3rd instar wild-type larvae. In a marked contrast, no TIM neurons were detected in the brain of 4-day-old 3rd instar npd -mutant larvae and the level of tim transcripts was approximately 10-fold lower in the NPD than in the wild-type strain. Daily changes in tim expression and TIM presence appeared to be under photoperiodic control in the wild-type larvae. Clear daily oscillations of tim transcription were observed during the development of 3rd instars under the short-day conditions. Daily oscillations were less apparent under the long-day conditions, where a gradual increase of tim transcript abundance appeared as a prevailing trend. Analysis of the genomic structure of tim gene revealed that npd-mutants carry a 1855 bp-long deletion in the 5'-UTR region. This deletion removed the start of transcription and promoter regulatory motifs E-box and TER-box. The authors hypothesize that this mutation was responsible for dramatic reduction of tim transcription rates, disruption of circadian clock function, and disruption of photoperiodic calendar function in npd-mutant larvae of C. costata.
Hop (Humulus lupulus L.), the essential source of beer flavor is of interest from a medicinal perspective in view of its high content in health-beneficial terpenophenolics including prenylflavonoids. The dissection of biosynthetic pathway(s) of these compounds in lupulin glands, as well as its regulation by transcription factors (TFs), is important for efficient biotechnological manipulation of the hop metabolome. TFs of the bZIP class were preselected from the hop transcriptome using a cDNA-AFLP approach and cloned from a cDNA library based on glandular tissue-enriched hop cones. The cloned TFs HlbZIP1A and HlbZIP2 have predicted molecular masses of 27.4 and 34.2 kDa, respectively, and both are similar to the group A3 bZIP TFs according to the composition of characteristic domains. While HlbZIP1A is rather neutral (pI 6.42), HlbZIP2 is strongly basic (pI 8.51). A truncated variant of HlbZIP1 (HlbZIP1B), which is strongly basic but lacks the leucine zipper domain, has also been cloned from hop. Similar to the previously cloned HlMyb3 from hop, both bZIP TFs show a highly specific expression in lupulin glands, although low expression was observed also in other tissues including roots and immature pollen. Comparative functional analyses of HlbZip1A, HlbZip2, and subvariants of HlMyb3 were performed in a transient expression system using Nicotiana benthamiana leaf coinfiltration with Agrobacterium tumefaciens strains bearing hop TFs and selected promoters fused to the GUS reference gene. Both hop bZIP TFs and HlMyb3 mainly activated the promoters of chalcone synthase chs_H1 and the newly cloned O-methyl transferase 1 genes, while the response of the valerophenone synthase promoter to the cloned hop TFs was very low. These analyses also showed that the cloned bZIP TFs are not strictly G-box-specific. HPLC analysis of secondary metabolites in infiltrated Petunia hybrida showed that both hop bZIP TFs interfere with the accumulation and the composition of flavonol glycosides, phenolic acids, and anthocyanins, suggesting the possibility of coregulating flavonoid biosynthetic pathways in hop glandular tissue.
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