Fractionation of heat-shocked Drosophila melanogaster Kc cells reveals that both the small heat shock proteins (hsp28, -26, -23, and -22) and vnmentin-like intermediate filament proteins (EFPs) are abundantly represented in the nuclear fraction. Cofractionation of the IFPs with nuclei is due to the collapse of the IFP network against the nucleus upon heat shock, raising the possibility that cofractionation of the small hsps is by a similar mehanis. Indirect Immunofluorescence supports this possibility. In salivary glands, both the hsps and the IFPs are cytoplasmic after mild-to-moderate heat shocks and only enter the nucleus upon severe-indeed, lethal-shocks. Double-label experiments with Schneider line 2 cells show that the IFPs and small hsps colocalize to the some perinuclear aggregates in 70% of the cells examined. Thus, the small hsps are associated with the cytoskeleton rather than with nuclear structures.The heat shock response is a ubiquitous cellular response to physiological stress characterized by the rapid induction of a small number of heat shock proteins (hsps; reviewed in refs. 1-3). The extreme conservation of the response and of the hsps themselves suggests that these proteins carry out essential functions. However, aside from their role in the development of thermotolerance-the ability to withstand a second heat shock at an otherwise lethal temperature (4-10)-little is known about their function(s). Thus far, the most progress has been made for hsp70. In yeast, replacement of several members of the hsp7O gene family with mutant genes indicates a role for hsp70 in growth at high temperatures (11). Likewise, mutants in dnaK (the Escherichia coli counterpart ofhsp70) are temperature-sensitive for growth (12) and are unable to recover normal protein synthesis following heat shock (13). hsp70 appears to play a similar role in Drosophila (14, 15) and apparently carries out this function within the nucleus (16).As a means of addressing the function of the low molecular weight family of hsps, several investigators have examined the intracellular distribution of these proteins. Several reports have argued for an association with chromatin and nucleoli (8,17). Yet, others appear to rule out association with chromatin and suggest that these proteins are part of the nuclear matrix (18,19 (20)(21)(22)(23). This cofractionation appears to be due to the collapse of the IFP network against the nucleus within 5 min of heat shock (20-23). In order to assess the contribution of the collapse of the IF cytoskeleton on the localization of the small hsps, we have reinvestigated the intracellular distribution of the small hsps ofDrosophila by biochemical fractionation and indirect immunofluorescence. The data best support association of these proteins with the IF cytoskeleton. For heat shock, cells were incubated at 370C for 15 min prior to harvest. Cells were rinsed with Echalier's medium, concentrated to 1 ml, and labeled with 50 ,uCi (1 Ci = 37 GBq) of [35S]methionine (Amersham) at 250C or 37TC for 1 hr. T...
A nearly universal feature of intron sequences is that even closely related species exhibit a large number of insertion/deletion differences. The goal of the analysis described here is to test whether the observed pattern of insertion/deletion events in the genealogy of the myosin alkali light chain (Mlc1) gene is consistent with neutrality, and if not, to determine the underlying forces of evolutionary change. Mlc1 pre-mRNA is alternatively spliced, and one constraint is that signals necessary for tissue-specificity of directed splicing must be conserved. If the total length of an intron is functionally constrained, then the distribution of indels on branches of the gene genealogy should reflect a departure from randomness. Here we perform a phylogenetic analysis, inferring ancestral states wherever possible on a phylogeny of 29 alleles of Mlc1 from six species of Drosophila. Observed patterns of indels on the genealogy were compared to those from simulated data, with the result that we cannot reject the null hypothesis of neutrality. A clear departure from a neutral prediction was seen in the excess folding free energy predicted for the introns flanking the alternatively spliced exon. Relative rate tests also suggest a retardation in the rate of Mlc1 sequence evolution in the simulans clade.
Interspecific comparisons of intron sequences reveal conserved blocks of invariant nucleotides and several other departures from the strictly neutral model of molecular evolution. To distinguish the past action of evolutionary forces in introns known to have regulatory information, we examined nucleotide sequence variation at 991 sites in a random sample of 16 Drosophila melanogaster alleles of the gene encoding the myosin alkali light chain (Mlc1). The Mlc1 gene of D. melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. Analyses of these data reveal that introns 4 and 5, which flank the alternatively spliced exon 5, have reduced levels of both intraspecific polymorphism and interspecific divergence relative to intron 3. No polymorphism was observed in any of the exons examined in D. melanogaster. A genealogical analysis clearly demonstrates the occurrence of intragenic recombination in the ancestral history of Mlc1. Recombination events are estimated to be 13 times more likely than mutation events over the span of the sequenced region. Although there is little evidence for pairwise linkage disequilibrium in the Mlc1 region, higher order disequilibrium does seem to be present in the 5' half of the portion of the gene that was examined. Predictions of the folding free energy of the pre-mRNA reveal that sampled alleles have a significantly higher (less stable) free energy than do randomly permuted sequences. These results are consistent with the hypothesis that introns surrounding an alternatively spliced exon are subjected to additional constraints, perhaps due to specific aspects of secondary structure required for appropriate splicing of the pre-mRNA molecule.
In an effort to (1) characterize the 67 interval of chromosome 3 of Drosophila melanogaster genetically and (2) isolate mutations of the 67B1 small heat shock protein (hsp) gene cluster specifically, we undertook a mutational analysis of the 67A-D subinterval. Using a deficiency of the 67A2 to 67D11-13 region, Df(3L)AC1, we screened 8700 diepoxybutane-treated chromosomes and 7800 ethyl methanesulfonate-treated chromosomes for visible and lethal mutations throughout this interval and recovered 74 independent recessive lethal mutations, but no visible mutations. One of the lethal mutations, d29A6, was identified as an overlapping deficiency extending from 66F3 to 67B1. An additional 6000 diepoxybutane-treated chromosomes were screened for lethality over d29A6, yielding another four lethal mutations within the 67A2-B1 subinterval. These 78 lethal mutations, along with two others isolated in other laboratories, define 23 essential loci--6 within the 67A2-B1 subinterval and 17 within the 67A2 to D11-13 subinterval. Many of these loci appear to be required for imaginal development only, exhibiting late larval to pharate adult lethal phases. Examination of the 67A2-B1 lethal complementation groups for (1) earlier onset of lethality following a heat shock, (2) missing or altered small hsps on two-dimensional protein gels, and (3) restoration of viability by transformed wild-type copies of the small hsp genes indicates that none of these mutations affect the small hsps. On the basis of this analysis and the known homology of the genes, we conclude that the small hsps are functionally equivalent.
The Mlcl gene of Drosophila melanogaster encodes two MLCl isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlcl pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlcl genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC 1 isoforms, suggests that exon 4 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis-regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlcl gene to maintain proper expression and function of the two MLCl polypeptides.
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