nalyot, a mutation of the Drosophila myb-related Adf1 transcription factor, disrupts synapse formation and olfactory memory.

nalyot (nal) is a novel olfactory memory mutant of Drosophila, encoding Adf1, a myb-related transcription factor. Following extended training sessions, Adf1 mutants show normal early memory but defective longterm memory. Adf1 shows widespread spatiotemporal expression, yet mutant alleles reveal no discernible disruptions in gross morphology of the nervous system. Studies at the larval neuromuscular junction, however, reveal a role for Adf1 in the modulation of synaptic growth-in contrast to the role established for dCREB2 in the control of synaptic function (Davis et al., 1996). These findings suggest that Adf1 and dCREB2 regulate distinct transcriptional cascades involved in terminal stages of synapse maturation. More generally, Adf1 provides a novel link between molecular mechanisms of developmental and behavioral plasticity.

Developmental expression of an amn(+) transgene rescues the mutant memory defect of amnesiac adults.

The Drosophila memory gene amnesiac (amn) has been proposed to encode a neuropeptide protein, which includes regions homologous to vertebrate pituitary adenylyl cyclase-activating peptide (PACAP; Feany and Quinn, 1995). Definitive experiments to link this gene to memory formation, however, have not yet been accomplished (Kandel and Abel, 1995). The experiments described here demonstrate that the putative amn transcript is involved in adult memory formation. With the use of a UAS-amn(+) transgene, we show complete rescue of memory defects in amn(28A), a mutant allele caused by the insertion of a GAL4 enhancer trap transposon (Moore et al., 1998). Study of the amn(28A) reporter reveals widespread expression in the adult brain but also enriched expression in the embryonic and larval nervous systems. To begin addressing the temporal requirement of amn in memory, we asked whether the memory defects could be rescued by restricting transgenic expression to the adult stage. A heat-shock regimen shown previously to rescue fully the amn ethanol sensitivity defect (Moore et al., 1998) failed to rescue the memory defect. These results, coupled with previous genetic and anatomical studies, suggest that adult memory formation and ethanol sensitivity have different temporal and spatial requirements for amn.

Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway.

Upon exposure to ethanol, Drosophila display behaviors that are similar to ethanol intoxication in rodents and humans. Using an inebriometer to measure ethanol-induced loss of postural control, we identified cheapdate, a mutant with enhanced sensitivity to ethanol. Genetic and molecular analyses revealed that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, we find that enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol. Taken together, our results provide functional evidence for the involvement of the cAMP signal transduction pathway in the behavioral response to intoxicating levels of ethanol.

Sequences regulating poly(A) site selection within the adenovirus major late transcription unit influence the interaction of constitutive processing factors with the pre-mRNA.

The adenovirus major late transcription unit (MLTU) encodes five families of mRNAs, L1 to L5, each distinguished by a unique poly(A) site. Use of the promoter-proximal L1 poly(A) site predominates during early infection, whereas poly(A) site choice shifts to the promoter-distal sites during late infection. A mini-MLTU containing only the L1 and L3 poly(A) sites has been shown to reproduce this processing switch. In vivo analysis has revealed that sequences extending 5' and 3' of the L1 core poly(A) site are required for efficient processing as well as for regulated expression. By replacement of the L1 core poly(A) site with that of the ground squirrel hepatitis virus poly(A) site, we now demonstrate that the L1 flanking sequences can enhance the processing of a heterologous poly(A). Upon recombination of the chimeric L1-ground squirrel hepatitis virus poly(A) site onto the viral chromosome, the L1 flanking sequences were also found to be sufficient to reproduce the processing switch during the course of viral infection. Subsequent in vitro analysis has shown that the L1 flanking sequences function to enhance the stability of binding of cleavage and polyadenylation specificity factor to the core poly(A) site. The impact of L1 flanking sequences on the binding of cleavage and polyadenylation specificity factor suggests that the regulation of the MLTU poly(A) site selection is mediated by the interaction of constitutive processing factors.

Dissection of memory formation: from behavioral pharmacology to molecular genetics.

Behavioral pharmacology has suggested an intricate, multiphasic pathway of memory consolidation. An integrated molecular pharmacological approach in Drosophila has lent support to this theory recently by dissecting consolidated memory into two genetically distinct components: a cycloheximide-insensitive, anesthesia-resistant memory and a cycloheximide-sensitive long-term memory. In addition, experiments using inducible dominant-negative transgenes in Drosophila or gene knockouts in mice demonstrate a role for cAMP-responsive transcription factors in formation of long-term memory. These studies support the application of reverse-genetic strategies, including the use of temporally specific agonists and antagonists, to advance the functional dissection of memory formation.

Relative roles of signals upstream of AAUAAA and promoter proximity in regulation of human immunodeficiency virus type 1 mRNA 3' end formation.

At least two mechanisms have been implicated in regulating poly(A) site use in human immunodeficiency virus type 1 (HIV-1): inhibition of basal signals within 500 nucleotides (nt) of the cap site, leading to specific suppression of the 5' poly(A) site, and stimulation of basal signals by long terminal repeat U3 sequences, leading to specific activation of the 3' poly(A) site. We determined the relative contributions of these mechanisms in a HeLa cell transcription/processing reaction and by transient transfection analysis. In vitro, the efficiency of basal signals is equivalent close to (270 nt) and far from (1,080 nt) the promoter and is stimulated at least 30-fold in both positions by upstream U3 sequences. In vivo, U3 sequences also enhance processing at both positions. There are two additional effects when the poly(A) site is close to the cap site: at least a 15-fold reduction in total RNA levels and a 5-fold decrease in relative levels of RNA processed at the HIV-1 site in constructs containing U3. Both effects are overcome by insertion of upstream splicing signals in an orientation-dependent manner. Splicing appears to influence poly(A)+ RNA levels by two distinct mechanisms: stabilizing nuclear transcripts and directly stimulating 3' end formation. It is proposed that upstream elements play major roles in regulating poly(A) site choice and in controlling the subsequent fate of polyadenylated RNA. The impact of these findings on mechanisms of mRNA biogenesis in the HIV-1 provirus is discussed.

Sequences regulating temporal poly(A) site switching in the adenovirus major late transcription unit.

Temporal regulation of poly(A) site choice occurs in an adenovirus recombinant encoding a miniature version of the major late transcription unit with two poly(A) sites, L1 and L3. Using deletion mutagenesis, we have looked directly for cis-acting elements regulating poly(A) site choice in this recombinant. From this work, we draw two main conclusions. First, elements other than the AAUAAA and downstream sequences of the L1 poly(A) site are required for temporal regulation of poly(A) site choice during infection. Second, these regions function in two distinct modes during infection. The two regions enhance selection of the L1 poly(A) site in an additive manner during an early infection, but deletion of either element abolishes the switch in poly(A) site choice during a late infection. This work documents the first example of a regulatory element downstream of a core poly(A) region.

Poly(A) site choice in retroelements: deja vu all over again?

The primary transcripts encoded by retroelements contain two polyadenylation [poly(A)] sites, positioned 5' and 3' of the protein-coding sequences. To ensure efficient gene expression in these systems, a mechanism must operate that suppresses use of the 5' site, enhances use of the 3' site, or both. These possibilities have been examined in a recent series of experiments that have revealed two main regulatory themes. First, maximum 3' end processing can require sequences other than core poly(A) signals, which are transcribed only before the 3' site. Second, processing is partly inhibited when the site is positioned close to the promoter. These results should serve as the basis for a more detailed understanding of how the choice of the poly(A) site is regulated in these elements.

Involvement of long terminal repeat U3 sequences overlapping the transcription control region in human immunodeficiency virus type 1 mRNA 3' end formation.

In retroviral proviruses, the poly(A) site is present in both long terminal repeats (LTRs) but used only in the 3' position. One mechanism to account for this selective poly(A) site usage is that LTR U3 sequences, transcribed only from the 3' poly(A) site, are required in the RNA for efficient processing. To test this possibility, mutations were made in the human immunodeficiency virus type 1 (HIV-1) U3 region and the mutated LTRs were inserted into simple and complex transcription units. HIV-1 poly(A) site usage was then quantitated by S1 nuclease analysis following transfection of each construct into human 293 cells. The results showed that U3 sequences confined to the transcription control region were required for efficient usage of the HIV-1 poly(A) site, even when it was placed 1.5 kb from the promoter. Although the roles of U3 in processing and transcription activation were separable, optimal 3' end formation was partly dependent on HIV-1 enhancer and SP1 binding site sequences.

Sequences upstream of AAUAAA influence poly(A) site selection in a complex transcription unit.

The adenovirus major late transcription unit (MLTU) encodes five colinear mRNA families, L1 through L5, each distinguished by a unique poly(A) site. Site selection is regulated during the course of infection, predominating early at the L1 site and late at the L2 through L5 sites. Two general mechanisms can be invoked to explain predominant usage of the L1 site early in infection. MLTU site selection may proceed in a first-come, first-serve manner whereby the L1 site is used most frequently because it is closest to the promoter. Alternatively, specific sequences flanking the L1 site may control predominant L1 site usage in a position-independent manner. To distinguish between these mechanisms, we constructed deletions in the L1 flanking sequences and inserted the mutated sites into either simple transcription units or mini-MLTUs encoding two poly(A) sites. The pattern of site selection for each construct was then quantitated by S1 nuclease analysis after transfection into 293 cells. The results indicated that L1 sequences upstream of AAUAAA define a novel selector element that can cause predominant L1 site usage at either position of a tandem transcription unit. The element did not significantly affect the stability or nucleocytoplasmic transport of L1 transcripts and was not required for efficient 3'-end processing in simple transcription units. Predominant L1 site usage required physical linkage of the processing signals and was independent of the major late promoter.

A herpes simplex virus type 1 mutant with a deletion in the polypeptide-coding sequences of the ICP4 gene.

A deletion mutant derived from herpes simplex virus type 1 (HSV-1) strain ANG was analysed. The deletion mapped within the polypeptide-coding region of the immediate-early ICP4 gene. Based on DNA sequence data the deletion was shown to comprise 84 base pairs. In the wild-type genome of strain ANG these sequences were almost completely homologous to the known sequences of HSV-1 strain 17. The ICP4 polypeptide induced by the mutant was similar in size to the wild-type ICP4 protein and was recognized by a monoclonal antibody against ICP4. The data presented suggest that the deletion corresponds to a region on the ICP4 polypeptide that is nonessential for the replication of the virus in vitro.