Transcriptional activators Cat8 and Sip4 discriminate between sequence variants of the carbon source-responsive promoter element in the yeast<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The structural genes for gluconeogenesis in the yeast Saccharomyces cerevisiae are activated by the carbon source-responsive element (CSRE) found in the respective upstream regions. Regulatory genes CAT8 and SIP4 both encode zinc-cluster proteins which can bind to CSRE motifs and activate target genes under conditions of glucose deprivation. In this work, we describe a functional analysis of sequence variants containing single mutations within the strongly activating CSRE(ICL1) motif. While the sequence CCNNNNNNCCG was required as the minimal UAS for gene activation by both Cat8 and Sip4, the activators responded differently to sequence variations in the central part of the CSRE. Our results allowed us to derive a consensus sequence for efficient gene activation by Cat8 (YCCNYTNRKCCG), while a more specific motif is required for activation by Sip4 (TCCATTSRTCCGR). Although their zinc cluster domains are clearly related, Cat8 and Sip4 are not isofunctional. This conclusion is further supported by the finding that biosynthetic derepression of Cat8 in the presence of a nonfermentable carbon source precedes that of Sip4 by about 90 min.     

Expression of a yeast gene can be blocked by insertion of short yeast DNA fragments between a UAS and the TATA box

We have constructed a plasmid, pOV10, which facilitates the introduction of putative upstream activating sequences (UAS) or upstream repressing sequences (URS) from yeast genes into plasmids containing CYC1-lacZ fusions. We have observed that the insertion of yeast sequences from 155 to 195 bp between the UAS and the TATA box of a CYC1-lacZ fusion gene can block -galactosidase expression. It is suggested that this block is related to the formation of nucleosomes on the DNA.     

Acute genetic manipulation of neuronal activity for the functional dissection of neural circuits-a dream come true for the pioneers of behavioral genetics

Abstract: This review summarizes technical development of the functional manipulation of specific neural circuits through genetic techniques in Drosophila. Long after pioneers' efforts for the genetic dissection of behavior using this organism as a model, analyses with acute activation of specific neural circuits have finally become feasible using transgenic Drosophila that expresses light-, heat-, or cold-activatable cation channels by xxx/upstream activation sequence (Gal4/UAS)-based induction system. This methodology opened a new avenue to dissect functions of neural circuits to make dreams of the pioneers into reality.     

Expression enhancement of the Tn5 neomycin-resistance gene by removal of upstream ATG sequences and its use for probing heterologous upstream activating sequences in yeast

We have constructed a series of promoter or upstream activating sequence (UAS)-probe plasmids carrying the Tn5-derived neomycin resistance gene whose seven additional ATG codons in the 5-untranslated region were completely or partially removed. When the deleted version of the neo sequence retaining only one additional ATG (NeoD) was expressed under the control of a TDH3 promoter whose UAS was deleted, the transformed cells were unable to grow at a low concentration of the antibiotic G418. In contrast with this, yeast cells expressing the NeoC sequence and having no additional ATG exhibited a high level of G418-resistance. Moreover, the UAS-probe system using NeoD has been successfully applied for the identification of several E. coli DNA sequences that clearly function as UASs in yeast cells. Two of these prokaryotic sequences with UAS activity were identified as a part of the coding region of the tgt and the hydG gene, respectively.