The suffix sequence is involved in processing the 3' ends of different mRNAs in Drosophila melanogaster

We describe here the nucleotide sequences of several genomic and mRNA copies of the suffix, a short dispersed actively transcribed repeat located at the 3' ends of many different genes of Drosophila melanogaster. Only one strand of the suffix is transcribed. The patterns of suffix-containing mRNAs vary during development. The five randomly selected genomic copies of the suffix are 265 bp long and quite conservative in their sequence. The non-transcribed strand is terminated with oligo(A) preceded by AATAAA sequence. No repetitive flanking sequences can be detected. The three other genomic copies selected by hybridization with suffix-containing cDNA clones are less conservative, especially in the 5' part. In particular, they contain short insertions carrying a polyadenylation signal AATAAA at exactly the same position of the suffix. Comparison of genomic and cDNA clones shows that mRNAs are polyadenylated at the last nucleotide of these insertions. The cDNA clones include the same part of the suffix, from the 39th to 112th nucleotide. Thus, a segment of the suffix forms the last exon for both genes. In one case, the beginning of the last intron coincides with the beginning of suffix, creating a very unusual donor splicing site. We conclude that the suffix sequence is directly involved in the formation of the last splicing site and 3'-end maturation of mRNA, at least in the case of the two genes analysed.     

Cloning of the 1.4-kb mRNA species of human complement factor H reveals a novel member of the short consensus repeat family related to the carboxy terminal of the classical 150-kDa molecule.

Three factor H mRNA species of 4.3 kb, 1.8 kb, and 1.4 kb are constitutively expressed in human liver. Having previously characterized full-length cDNA clones derived from the 4.3-kb and 1.8-kb factor mRNA, we report here the isolation and eucaryotic expression of full-length cDNA clones coding for the 1.4-kb mRNA species. The 1266-bp cDNA codes for a polypeptide of 330 amino acids and contains two polyadenylation signals and a short poly(A)+tail. The protein is composed of a leader peptide followed by five short consensus repeat domains. It shows a hybrid structure with the last three domains being almost identical to the carboxy-terminal of the classical 150-kDa factor H molecule and the two first domains representing unique short consensus repeat structures. Eucaryotic expression in COS7 cells revealed two polypeptides derived from one cDNA clone that are also found in human serum. Differences between the cDNA clones within the last three domains indicate two distinct, possibly allelic sequences that, in addition, differ from the authentic 150-kDa factor H sequence. Southern blot results support the notion that the 4.3-kb factor H and the 1.4-kb factor H-related mRNA are transcribed from two separate but highly homologous genes.     

Lack of in vivo transcription of Acetabularia mediterranea 1175 bp ctDNA fragment homologous to the Drosophila per locus.

The period (per) locus of Drosophila melanogaster has a fundamental role in the expression of biological rhythms. A DNA sequence homologous to a short region of the Drosophila per locus was detected in the chloroplast of Acetabularia mediterranea. A 1175 bp DNA fragment containing the sequence was used as a probe in 'Northern' hybridization experiments. It was found that this DNA was not transcribed or only marginally transcribed in A. mediterranea, at least at the developmental stage just prior to cap formation. It seems that the 1175 bp ctDNA fragment is not involved in the Acetabularia biological rhythm mechanism.     

Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA

We report the cloning, nucleotide (nt) sequence and expression of the cDNA (pah) encoding phenylalanine hydroxylase (PAH) of Drosophila melanogaster. The strong hybridization signals observed in genomic blots when D. melanogaster DNA was probed with 32P-labeled human pah cDNA, indicated the existence of a high degree of sequence similarity between the pah genes of both species. The length of the pah genomic fragment is about 30 to 40 kb. The cDNA contains 84 bp of the 5'-untranslated region, 1359 bp of the protein-coding region and 87 bp of the 3' region, with only one polyadenylation signal. The isolated cDNA is probably full-length, since the size of the D. melanogaster PAH mRNA is 1.5 kb. At the nt level, the similarity of the D. melanogaster cDNA with human and rat pah cDNAs is 57.9% and 58.1%, respectively. The highest similarities are restricted to the nt sequence coding for the presumed hydroxylation domain. There is no nt sequence similarity between the first three exons of the human pah gene and an equivalent fraction of the D. melanogaster pah gene. At the amino acid (aa) level, the similarity in the presumed hydroxylation domain is 88.5%, in which two motifs of the structure AGLLSSXXXL are found, where X represents any aa. It was interesting to notice the conservation of aa 408, 311 and 280, where mutations are associated with phenylketonuria in humans. We observed, moreover, that, as it occurs in humans and rats, the expression of the D. melanogaster pah gene is tissue-specific and temporally regulated.     

Control of poly(A) polymerase level is essential to cytoplasmic polyadenylation and early development in Drosophila

Poly(A) polymerase (PAP) has a role in two processes, polyadenylation of mRNA precursors in the nucleus and translational control of certain mRNAs by cytoplasmic elongation of their poly(A) tails, particularly during early development. It was found recently that at least three different PAP genes exist in mammals, encoding several PAP isoforms. The in vivo specificity of function of each PAP isoform currently is unknown. Here, we analyse PAP function in Drosophila: We show that a single PAP isoform exists in Drosophila that is encoded by the hiiragi gene. This single Drosophila PAP is active in specific polyadenylation in vitro and is involved in both nuclear and cytoplasmic polyadenylation in vivo. Therefore, the same PAP can be responsible for both processes. In addition, in vivo overexpression of PAP does not affect poly(A) tail length during nuclear polyadenylation, but leads to a dramatic elongation of poly(A) tails and a loss of specificity during cytoplasmic polyadenylation, resulting in embryonic lethality. This demonstrates that regulation of the PAP level is essential for controlled cytoplasmic polyadenylation and early development.     

Ribosomal protein insufficiency and the minute syndrome in Drosophila: a dose-response relationship.

Minutes comprise > 50 phenotypically similar mutations scattered throughout the genome of Drosophila, many of which are identified as mutations in ribosomal protein (rp) genes. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. By mobilizing a P element inserted in the 5'' UTR of M(3)95A, the gene encoding ribosomal protein S3 (RPS3), we have generated two homozygous viable heteroalleles that are partial revertants with respect to the Minute phenotype. Molecular characterization revealed both alleles to be imprecise excisions, leaving 40 and 110 bp, respectively, at the P-element insertion site. The weaker allele (40 bp insert) is associated with a approximately 15% decrease in RPS3 mRNA abundance and displays a moderate Minute phenotype. In the stronger allele (110 bp insert) RPS3 mRNA levels are reduced by approximately 60%, resulting in an extreme Minute phenotype that includes many morphological abnormalities as well as sterility in both males and females due to disruption of early gametogenesis. The results show that there is a correlation between reduced RPS3 mRNA levels and the severity of the Minute phenotype, in which faulty differentiation of somatic tissues and arrest of gametogenesis represent the extreme case. That heteroalleles in M(3)95A can mimic the phenotypic variations that exist between different Minute/rp-gene mutations strongly suggests that all phenotypes primarily are caused by reductions in maximum protein synthesis rates, but that the sensitivity for reduced levels of the individual rp-gene products is different.     

Isolation of cDNA clone encoding rat senescence marker protein-30 (SMP30) and its tissue distribution.

We have isolated and characterized two cDNA clones encoding senescence marker protein-30 (SMP30), the amounts of which are known to decrease androgen-independently with aging in the livers of rats. Of these cDNA clones, one consisted of 1588 bp nucleotides and the other of 1195 bp nucleotides generated by alternative polyadenylation. These two cDNA clones shared the same open reading frame, but the larger species had 393 bp nucleotides of 3' untranslated region in addition to the first polyadenylation site of smaller species. Northern hybridization analysis showed that two species of mRNA (1.7 kb and 1.4 kb) located in the liver and kidney were consistent with these short and long forms of cDNA. The open reading frame, 897 bp could encode 299 amino acids. The estimated molecular weight and pI of the deduced polypeptide were 33,387 and 5.1, respectively. Furthermore, immunohistochemical analysis confirmed that SMP30 was preferentially localized in the hepatocytes and renal proximal tubular epithelium. Genomic Southern hybridization analysis demonstrated that SMP30 was widely conserved among higher animals. A computer-assisted homology analysis of nucleic acid and protein databases revealed no remarkable homology with other known proteins. Therefore, SMP30 seems to be a novel protein. In addition, the existence of putative A-U rich mRNA degradation signals and protein degradation signals (PEST sequence) in the structure of SMP30 may suggest important regulatory function of this unique protein manifested by changes in its concentrations.     

Molecular cloning and the nucleotide sequence of cDNA to mRNA for non-neuronal enolase (alpha alpha enolase) of rat brain and liver.

The nucleotide sequence for alpha alpha enolase (non-neuronal enolase: NNE) of rat brain and liver was determined from recombinant cDNA clones. The sequence was composed of 1722 bp which included the 1299 bp of the complete coding region, the 108 bp of the 5'-noncoding region and the 312 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, the poly(A) tail was also found. A potential ribosome-binding site was located 30 nucleotides upstream to the initiation codon in the 5'-noncoding region. The amino acid sequence deduced from the nucleotide sequence was 433 amino acids in length and showed very high homology (82%) to the amino acid sequence of gamma gamma enolase (neuron-specific enolase: NSE), although the nucleotide sequence showed slightly lower homology (75%). The size of NNE mRNA was approximately 1800 bases by Northern transfer analysis and much shorter than that of NSE mRNA (2400 bases) indicating a short 3'-noncoding region. A dot-blot hybridization and Northern transfer analysis of cytoplasmic RNA from the developing rat brains using a labeled 3'-noncoding region of cDNA (no homology between NSE and NNE) showed a decrease of NNE mRNA at around 10 postnatal days and then a gradual increase to adult age without changes of mRNA size. Liver mRNA did not show any significant change during development.     

Cooperative enhancement at the Drosophila Sgs-3 locus

The Drosophila glue gene Sgs-3 is specifically expressed in the secretory cells of the salivary glands of third instar larvae. We have assayed the expression of gene fusions to determine the role of cis-acting Sgs-3 sequences in conferring this pattern of expression. These experiments define two regulatory regions required for expression of reporter genes from the Sgs-3 promoter. One region, between 106 and 56 bp upstream of the Sgs-3 mRNA 5' end is sufficient for low but correct tissue- and stage-specific expression. A second region, lying between 629 and 130 bp 5' of the RNA start site is functionally equivalent; that is, it alone will also direct low level, specific expression. These two regions act synergistically to give high level expression. More distant upstream regions function to further increase levels of expression. These two regulatory elements can confer a salivary gland-specific pattern of expression on a heterologous promoter and are also sufficient to drive gene expression in other Drosophila species, implying conservation of regulators.     

Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.