Nuclear pre-mRNA metabolism: channels and tracks.

We review new evidence suggesting that metazoan nuclear pre-mRNA metabolism occurs in a small subnuclear compartment consisting of a network of channels defined by exclusion from various condensed structures. Nuclear components, including mRNA en route from the gene to the nuclear surface, apparently move through these channels by conventional diffusion.

Evidence for channeled diffusion of pre-mRNAs during nuclear RNA transport in metazoans.

We report studies using an enhanced experimental system to investigate organization of nuclear pre-mRNA metabolism. It is based on the powerful genetic system and polytene nuclei of Drosophila. We observe (at steady state) movement of a specific pre-mRNA between its gene and the nuclear surface. This movement is isotropic, at rates consistent with diffusion and is restricted to a small nuclear subcompartment defined by exclusion from chromosome axes and the nucleolus. Bulk polyadenylated nuclear pre-mRNA precisely localizes in this same subcompartment indicating that most or all pre-mRNAs use the same route of intranuclear movement. In addition to association with nascent transcripts, snRNPs are coconcentrated with pre-mRNA in this subcompartment. In contrast to constitutive splices, at least one regulated splice occurs slowly and may undergo execution remotely from the site of pre-mRNA synthesis. Details of our results suggest that retention of incompletely spliced pre-mRNA is a function of the nuclear surface. We propose a simple model--based on channeled diffusion--for organization of intranuclear transport and metabolism of pre-mRNAs in polytene nuclei. We argue that this model can be generalized to all metazoan nuclei.

Molecular cloning and genetic analysis of the suppressor-of-white-apricot locus from Drosophila melanogaster.

We report genetic and molecular analysis of the suppressor-of-white-apricot [su(wa)] locus, one of several retrotransposon insertion allele-specific suppressor loci in Drosophila melanogaster. First, we isolated and characterized eight new mutations allelic to the original su(wa)1 mutation. These studies demonstrated that su(wa) mutations allelic to su(wa)1 affected a conventional D. melanogaster complementation group. Second, we cloned the chromosomal region containing the su(wa) complementation group by P element transposon tagging. The ca. 14-kilobase region surrounding the su(wa) complementation group contained five distinct transcription units, each with a different developmentally programmed pattern of expression. Third, we used a modified procedure for P-mediated gene transfer to identify the transcription unit corresponding to su(wa) by gene transfer. Fourth, we found that the presumptive su(wa) transcription unit produced a family of transcripts (ranging from ca. 3.5 to ca. 5.2 kilobases) in all developmental stages, tissue fractions, and cell lines we examined, suggesting that the gene is universally expressed.

A detailed developmental and structural study of the transcriptional effects of insertion of the Copia transposon into the white locus of Drosophila melanogaster.

The copia insertion responsible for the wa mutation is 3' to the white promotor and in the same transcriptional orientation as white. First, we have analyzed the effects of the wa copia insertion on levels of polyadenylated white transcripts and find large, developmentally programmed effects. Second, we have isolated and sequenced an LTR-excision event involving the copia insertion at wa. This represents the first documented case of an LTR-excision event in Drosophila. This single copia LTR has developmentally programmed effects on white transcript levels qualitatively similar to the intact copia element. Third, we have characterized the structures of white transcripts from wa. We find polyadenylated white transcripts apparently having 3' termini in or near the 3' LTR of the wa copia insertion, as has been reported in limited studies of wa transcription in adults by others. These earlier studies also revealed wa transcripts apparently corresponding to polyadenylated terminus formation in the 5' LTR of the copia transposon; however, our more detailed studies reveal that these transcripts probably have other origins and that little, if any, polyadenylated terminus formation for white transcripts occurs in the 5' LTR of the wa copia insertion. Moreover, we find no polyadenylated terminus formation for white transcripts occurring in the single LTR of the wa LTR-excision product. Fourth, we find that each of three mutant alleles at su(wa) produces elevated levels of several classes of RNAs apparently corresponding to transcriptional readthrough of the wa copia transposon. Elevated levels of one presumptive readthrough transcript were observed previously in one su(wa) mutant strain. Fifth, we have confirmed the existence of a transcript initiated in the 3' LTR of the wa copia insertion and find the levels of this transcript to be strongly influenced by developmental stage and genetic background. Lastly, we have analyzed white transcripts produced by the whd81b11 allele, which carries an insertion of copia in the opposite transcriptional orientation and in a different position than the wa copia insertion. In contrast to the wa copia insertion allele, the whd81b11 allele produces polyadenylated white transcript levels very similar to the w+ case at the stages examined. Moreover, the whd81b11 copia element apparently produced polyadenylated terminus formation in white transcripts and we observe no effect of the allelic state of su(wa) on apparent readthrough of this stop site.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of autoregulation at the level of pre-mRNA splicing of the suppressor-of-white-apricot gene in Drosophila.

The Drosophila suppressor-of-white-apricot [su(wa)] protein regulates/modulates at least two somatic RNA processing events. It is a potent regulator of its own expression. We report here new studies of this autoregulatory circuit. Among other things, our studies show the following. First, new evidence that su(wa) expression is autoregulated at the level of pre-mRNA splicing is reported. su(wa) protein represses accumulation of the fully spliced su(wa) mRNA encoding it and promotes accumulation of high levels of incompletely spliced su(wa) pre-mRNA. Second, the fully spliced su(wa) mRNA is sufficient for all known su(wa) genetic functions indicating that it encodes the sole su(wa) protein. Third, the incompletely spliced su(wa) pre-mRNAs resulting from autoregulation are not translated (probably as a result of nuclear retention) and apparently represent nonfunctional by-products. Fourth, the special circumstances of su(wa) expression during oogenesis allows maternal deposition exclusively of fully spliced su(wa) mRNA. Fifth, su(wa) protein immunolocalizes to nuclei consistent with its being a direct regulator of pre-mRNA processing. We discuss the implications of our results for mechanisms of splicing regulation and for developmental control of su(wa) expression.

Evidence that two mutations, wDZL and z1, affecting synapsis-dependent genetic behavior of white are transcriptional regulatory mutations.

Both the white mutant allele, wDZL, and the zeste mutant allele, z1, reduce white transcript levels in adult head tissues, but have no effect on these levels in other tested adult tissues. wDZL results from insertion of a complex transposon approximately 5.0 kb 5' to white. Juxtaposition of this transposon to white produces a novel transcription unit beginning in the transposon and producing a mature transcript containing both white and transposon sequences. Head-specific expression of this novel transcription unit correlates with the observed repression of wild-type white transcript levels. wDZL and z1 are known to cause synapsis-dependent repression of w+ expression in trans as assessed by eye pigment deposition. Collectively, these results suggest an unexpected and informative correspondence between cis and synapsis-dependent trans regulatory effects. We discuss the implied mechanistic relationship between regulation of transcription and transvection effects in Drosophila.

Microbial interference and colonization of the murine gastrointestinal tract by Listeria monocytogenes.

Two strains of Listeria monocytogenes, one that formed smooth colonies on agar surfaces and a varient of it that formed rough colonies, colonized the gastrointestinal tracts of germfree mice. Within 24 h after mice were inoculated orally with about 100 bacteria, the population levels per gram (wet weight) of tissue of both strains were 10(5) to 10(7) in the stomach and ileum and 10(8) to 10(9) in the cecum and colon, respectively. As detected in Gram-stained histological sections, in such gnotobiotes, the bacteria colonized the lumen in all areas of the tract and much of the mucus layer on the epithelial surface in the proximal colon. The strain that formed smooth colonies did not colonize the tracts of specific-pathogen-free mice, but did colonize, to the same levels as in germfree mice, the stomachs and bowels of ex-germfree mice previously associated with two members of the indigenous flora (Bacteroides and Clostridium). In the latter animals, however, the listeria did not form layers on the colonic epithelium as efficiently as they did in monoassociated gnotobiotes.

Regulation of white locus expression: the structure of mutant alleles at the white locus of Drosophila melanogaster.

We have analyzed the structures of 19 mutant alleles at the white locus of Drosophila melanogaster. Thirteen of the mutant alleles in our selected sample arose spontaneously, and of these, seven are associated with insertions of non-white-region DNA sequence elements. Several lines of evidence strongly suggest that these insertions are responsible for their associated mutant alleles, and further suggest that most or all of these insertions are transposons. Moreover, the white locus DNA sequences can be divided into two nonoverlapping domains on the basis of the properties of the two domains as mutational targets. One of these domains behaves, in this regard, in the manner expected of functional coding sequences, whereas the other does not. We propose a model for the nature and function of the presumptive noncoding white locus genetic elements. The two domains of the white locus defined by our studies are approximately coextensive with the functionally distinct subintervals of the locus defined by previous genetic analysis. Lastly, our results strongly suggest that the dominant, mutable wDZL allele results from the insertion of a transposon outside of, but near, the white locus. This putative transposon apparently carries genetic elements that act at a distance to repress expression of the white locus.

Concanavalin A-mediated affinity film for Treponema pallidum.

Freshly extracted Treponema pallidum bound to glass cover slips preexposed to specific lectins, permitting biological testing in the absence of complex tissue fluids.

Developmental expression of a regulatory gene is programmed at the level of splicing.

We report sequence and transcript structures for a 6191-base chromosomal segment containing the presumptive regulatory gene from Drosophila, suppressor-of-white-apricot [su(wa)]. Our results indicate that su(wa) expression is controlled by regulating occurrence of specific splices. Seven introns are removed from the su(wa) primary transcript during precellular blastoderm development. The sequence of this mature RNA indicates that it is a conventional messenger RNA. In contrast, after cellular blastoderm the first two of these introns cease to be efficiently removed. The mature RNAs resulting from this failure to remove the first two introns have structures quite unexpected of mRNAs. We propose that postcellular blastoderm su(wa) expression is repressed by preventing splices necessary to produce a functional mRNA. Implications and mechanisms are discussed.

Evidence that a regulatory gene autoregulates splicing of its transcript.

Expression of the presumptive regulatory gene, suppressor-of-white-apricot [su(wa)], is controlled at the level of splicing. Results reported here indicate that this control represents autorepression of su(wa) expression. Specifically, reverse genetic studies demonstrate that the 3.5 kb mature su(wa) RNA (produced by removal of seven introns) is a message essential for su(wa)+ function and indicate that the abundant 4.4 kb and 5.2 kb mature su(wa) RNAs (resulting when the first or first and second of the seven introns are not removed) are, unexpectedly, byproducts of repression of production of the functional 3.5 kb RNA. Moreover, several experiments indicate that this repression of splices necessary to produce the 3.5 kb RNA is dependent on the translation product of the 3.5 kb RNA itself. We propose that this regulatory gene autoregulates its expression by controlling splicing of its primary transcript.