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PubMed2006

Isolation and characterization of another cDNA encoding a chorismate mutase from the phytoparasitic nematode Meloidogyne arenaria.

A new cDNA, named Ma-cm-2, encoding a chorismate mutase (CM), has been isolated from Meloidogyne arenaria. The full-length cDNA, carrying the trans-spliced SL1 leader sequence, was 753-bp long with an open reading frame of 576 bp. The deduced protein MA-CM-2 including amino-terminal signal peptide shows significant similarity to CMs of Meloidogyne incognita, Meloidogyne javanica, and also bacteria. Secondary structure prediction of MA-CM-2 indicates the presence of the three conserved alpha-helix domains present in the Escherichia coli CMs. Reverse transcription and polymerase chain reaction analysis showed that its transcript abundance is high in the early developmental stages and low in later ones. In situ mRNA hybridization revealed that the transcripts of Ma-cm-2 accumulated specifically in the two subventral oesophageal gland cells of M. arenaria. The widespread existence of CMs in the sedentary endoparasitic nematodes implicates that this enzyme plays an important role in the host-parasite interaction.

Amino Acid Sequence↗

PubMed1984

The junctions between the repetitive and the short unique sequences of the herpes simplex virus genome are determined by the polypeptide-coding regions of two spliced immediate-early mRNAs.

Immediate-early (IE) mRNAs-4 and -5 of herpes simplex virus type 2 (HSV-2) are transcribed from the IRs/TRs genome regions towards the Us region. Each of these spliced mRNAs has an untranslated leader sequence of 249 bases and a single intron of approximately 540 bases which are contained entirely within TRs/IRs sequences. The DNA sequence of the intron largely comprises tandem reiterations of three distinct short sequences. Upstream of the common 5' mRNA termini the DNA sequence contains regions of homology with the equivalent region of HSV-1. Comparison of the polypeptides encoded by these HSV-2 mRNAs with those of HSV-1 shows blocks of conserved amino acids. The locations of the first initiator ATG triplets of these two HSV-2 mRNAs suggest that the IRs/TRs regions of HSV expand, by gene conversion or by equal though non-homologous crossover, to an extent determined by the functions of the DNA sequences which are duplicated or deleted as a result of the crossover. This mechanism for expansion of repeats may apply to other herpesviruses which have a genome structure similar to that of HSV.

Animals↗

PubMed1995

The translational repression mediated by the platelet-derived growth factor 2/c-sis mRNA leader is relieved during megakaryocytic differentiation.

Expression of the platelet-derived growth factor 2/c-sis gene is highly restricted and controlled at multiple levels. Its structured mRNA leader, which is unusually long (1022 nucleotides), serves as a potent translational inhibitor. One of the sites of PDGF2 synthesis is megakaryocytes, implying that PDGF2 translation efficiency is modulated during megakaryocytic differentiation. To study the role of the mRNA leader as a translational cis-modulator, the hybrid T7/vaccinia cytoplasmic expression system was used to disconnect between determinants controlling transcription, alternative splicing, and mRNA stability from those controlling translation. Chimeric transcripts in which the human PDGF2/c-sis mRNA leader positioned in frame upstream of a reporter gene were used to determine whether the mRNA leader can confer variable translational efficiencies during differentiation. It is demonstrated that there is a time window during megakaryocytic differentiation of K562 cells in which the strong translational inhibition by PDGF2/c-sis mRNA leader is relieved. The time course of the translational repression relief is similar to that of PDGF2/c-sis transcriptional induction during the differentiation process. A 179-nucleotides CG-rich fragment immediately upstream of the initiator AUG codon is necessary for coffering stringent modulation of the translational efficiency. In NIH3T3 overexpressing translation initiation factor eIF4E, the inhibitory effect of the mRNA leader of c-sis is not relieved, suggesting that the changes in the translational machinery during megakaryocytic differentiation are beyond eIF4E activity. The possible involvement of a 5'-end-independent translational mechanism is discussed.

Base Sequence↗

PubMed1989

Multiple polyadenylation sites in a large 3'-most exon of the rat insulin-like growth factor II gene.

The rat insulin-like growth factor II (rIGFII) gene produces, in addition to three major mRNA species 3.6 kilobases (kb), 4.6 kb and 3.8 kb in length which represent transcripts from three independent leader-exons, multiple smaller-sized products that distribute broadly in the 1-3 kb region on Northern blots. Structural constituents of these RNAs were analyzed by hybridization with region-specific probes prepared from the entire rIGFII genome. Most of these shorter RNAs contained both 5'-untranslated and coding regions, but only parts of the 3'-untranslated region. At least nine protected sites were mapped within a single 3'-most exon E6 by S1 nuclease analysis. Some but not all of these sites were associated with the upstream polyadenylation signal, AATAAA, or its variants. Since none of the shorter subspecies contained intronic sequences, aberration in splicing is not involved in their generation. Thus, the main parts of submature materials are a collection of discrete species of RNAs, most, if not all, of which are produced by alternative polyadenylation site selection.

Amino Acid Sequence↗

PubMed2000

Homozygous HLA-G*0105N healthy individuals indicate that membrane-anchored HLA-G1 molecule is not necessary for survival.

HLA-G is expected to play an important role during fetal development. Recently, a healthy individual homozygous for the HLA-G*0105N allele has been described, suggesting that HLA-G expression was not essential for fetal survival. We now report studies of one family with five healthy siblings homozygous for HLA-G*0105N, who had been normally delivered; three of these siblings were females who also had normal deliveries. In addition, HLA-G*0105N cDNA has been fully sequenced, and normal G1 membrane anchored protein cannot be translated since after the codon 130 cytosine deletion (exon 3) a reading frameshift is observed leading to the existence of premature stop codon at position 189 (beginning of exon 4). Other protein isoforms (G2, G3 and G6), all containing the leader peptide and the alpha1 domain, are possible and their messenger mRNAs were found; any of these may undertake the necessary HLA-G functions. Our data show that the membrane anchored HLA-G molecule is not necessary in either mother or fetus for a normal pregnancy and survival. Also, individuals homozygous for HLA-G*0105N are healthy and with no indications of immunodeficiency or autoimmunity.

Alleles↗

PubMed1998

Molecular analysis of PHYA in wild-type and phytochrome A-deficient mutants of tomato.

Tomato (Lycopersicon esculentum Mill., recently redesignated Solanum lycopersicum L.), an agronomically important crop plant, has been adopted as a model species complementary to Arabidopsis in which to characterize the phytochrome family. Here we describe the cloning and molecular characterization of the gene encoding the apoprotein of phytochrome A in wild-type tomato and in the far-red-light-insensitive (fri1 and fri2) tomato mutants. The physical organization of this gene is similar to that of other angiosperm phytochromes with the four exons of the coding region interrupted by three introns. The pool of transcripts is heterogeneous due to multiple transcription start sites and to three modes of alternative splicing of the 5' leader. The leader in each alternative transcript carries multiple upstream open reading frames of considerable length. At the genomic level, both fri mutants share an identical base substitution which changes a consensus AG/ to TG/ at the 3' end of the intron between exons 1 and 2. This mutation leads to aberrant processing of the resultant pre-mRNA. While most mature transcripts retain the mutated intron, both cryptic splicing and exon skipping were also detected. Cryptic splicing occurred both upstream and downstream from the wild-type splice site. These observations are consistent with the hypothesis that exon definition in splicing of plant pre-mRNAs plays a secondary role to that of intron definition. Analysis of the frequency with which potentially functional phytochrome A apoproteins might be produced indicates that both fri1 and fri2 have less than 1% of the wild-type phytochrome A level.

Alternative Splicing↗

PubMed1995

The amylose content in rice endosperm is related to the post-transcriptional regulation of the waxy gene.

The waxy (Wx) gene of rice encodes a granule-bound starch synthase (GBSS = waxy protein) required for the synthesis of amylose in endosperm. An analysis of Wx transcripts, Wx protein, and amylose content of 31 rice cultivars revealed that endosperm amylose and Wx protein contents are correlated with the ability of the cultivar to excise intron I from the leader sequence of the Wx transcript. Cultivars with high endosperm amylose content (group I) contain high levels of amylose, Wx protein, and the mature 2.3 kb Wx mRNA. Cultivars with intermediate amylose content (group II) produce substantial amounts of a large 3.3 kb Wx pre-mRNA, with intron I still present, in addition to the mature Wx mRNA, and intermediate levels of Wx protein. Glutinous rice (group III cultivars) contains no amylose, no Wx protein, and no mature Wx mRNA; only the incompletely spliced 3.3 kb Wx pre-mRNA is present in group III cultivars. Based on these results, it is hypothesized that the amylose content of rice endosperm is regulated at the level of Wx transcript processing, and, more specifically, at the stage of intron I excision from the Wx pre-mRNA.

Amino Acid Sequence↗

PubMed1999

Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast.

cDNAs showing high sequence similarity (>70%) over large stretches to plant CYP73A orthologues from other species were isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. These clones appear to code for a full-length 1554 bp open reading frame with a 78 bp 5'-untranslated region and a 140 bp 3'-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with a predicted Mr of 59229 and a pI of 8.8. It contains the conserved cysteine haem-binding site found in all cytochrome P450s. The protein encoded by this cDNA diverges however from other CYP73As in its N- and C-terminus and in four domains internally, so that overall sequence similarity is in the range 58-66%. Many clones contained an identical intron, which may be associated with a novel regulatory mechanism. Sequence similarity is sufficient for it to be classified as CYP73A15, although it is the least similar member of this family classified so far. The cDNA was expressed in yeast. Successful expression of cinnamate 4-hydroxylase activity required removal of the intron. High-level expression also required modification of the N-terminus to that of CYP73A1. Yeast did not process the intron at all and the leader sequence for A15 was not as compatible as that of A1. The mRNA for CYP73A15 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean but induction was more transient than that of phenylalanine ammonia-lyase (PAL). In contrast, induction in cells undergoing xylogenesis was much more coordinate with PAL. The cloned cDNA may represent a cinnamate 4-hydroxylase isoform, whose expression is more related to differentiation than the responses to stress in which the majority of CYP73As cloned so far are involved.

Amino Acid Sequence↗

PubMed1991

Structure of the mouse beta Fc gamma receptor II gene.

We have isolated and deduced the structure of the beta Fc gamma RII gene from the mouse. This gene spans approximately 18 kb of DNA; the structure and nucleotide sequence has been determined and potential regulatory sites identified. This gene is composed of 10 exons that give rise to the beta 1 and beta 2 Fc gamma RII cDNA. Four exons encode the 5' untranslated region and leader sequence, one exon for each Ig-binding domain and membrane-spanning region and three exons encoding the cytoplasmic tail and 3' untranslated region. Analysis of the gene structure indicated that the beta 1Fc gamma RII and beta 2Fc gamma RII arise by differential mRNA splicing when a single 141-bp exon (exon 8) is alternately spliced to give rise to these isoforms. Sequence analysis of 2 kbp upstream of the transcription start site indicated the presence of regulatory elements, including three binding sites for the transcription factor Sp1, and Ap-4 binding site, a tandem glucocorticoid response element, and an overlapping tandem repeat. Furthermore, as in the Thy-1 gene, no CAT or TATA consensus sequences were observed. In addition, sites of methylation that regulate expression of this gene were also located at the 5' end of the gene and within several introns. A large intron located between exons 4 and 5 also contained a series of purine/pyrimidine-rich regions and a potential enhancer sequence.

Amino Acid Sequence↗

PubMed2000

The spliceosomal intron of the rolA gene of agrobacterium rhizogenes is a prokaryotic promoter.

Agrobacterium rhizogenes transfers DNA (T-DNA) from its Ri plasmid to plant cells. All T-DNA genes are expressed in plant cells. The rolA gene is the only T-DNA gene that contains an intron in the untranslated leader region of its mRNA. This paper shows that (i) the rolA gene is also transcribed in bacteria; (ii) the 85 bp corresponding to the spliceosomal intron drives prokaryotic gene expression in agrobacteria, in free-living rhizobia and in bacteroids within root nodules; and (iii) promoter activity is abolished by the deletion of 63 bp from its 5' end and is reduced by mutations changing its sequence near the putative -10 region. The expression pattern of a chimeric reporter gene shows that, in free-living bacteria, gene expression takes place during the exponential phase of growth and increases at the onset of the stationary phase. Within root nodules, reporter gene expression occurs in the invasion, nitrogen fixing and senescent zones.

Bacterial Proteins↗

PubMed1981

Unspliced functional late 19S mRNAs containing intervening sequences are produced by a late leader mutant of simian virus 40.

dl-805 is a viable deletion mutant of simian virus 40 lacking 187 of the 202 nucleotides coding for the principal leader of the wild-type viral late mRNAs. The structures of the mutant late 16S and 19S RNAs and proteins were examined with the following principal findings. (i) The vast majority, if not all, of the 19S RNAs are unspliced over their entire length. These RNAs contain intervening sequences and serve as mRNAs. The vast majority, if not all, of the 16S RNAs, however, retain the characteristic wild-type splice. (ii) Mutant 16S and 19S RNAs do not terminate at residue 243 (located six nucleotides upstream from the 5' end of the deleted DNA segment), the principal 5' terminus of the wild-type late mRNAs; rather, they begin at a series of sites, mostly upstream, used infrequently or not at all by wild-type virus. (iii) Despite the presence of altered late mRNAs, dl-805 late proteins have electrophoretic mobilities similar to those of wild-type late proteins, and mutant virions contain the same relative quantities of late proteins as wild-type virions.

Base Sequence↗

PubMed1984

Characterization of the major mRNAs from adenovirus 2 early region 4 by cDNA cloning and sequencing.

The sequences at the splice junctions of many early region 4 (E4) mRNAs from adenovirus 2 (Ad2) were determined by analysis of cDNA clones. The cDNAs were synthesized from poly(A)+ mRNA isolated from HeLa cells early during Ad2 infection. A standard library was constructed, in pBR322, from double stranded cDNAs initiated by oligo-dT priming. Approximately 1% of total recombinants contained E4 sequences, however among eighty clones analyzed in detail, only four contained the 5' leader sequence. A second library was prepared using a new method that led to a greatly increased representation of desired clones. This method employed oligo-dT to prime the synthesis of the first strand and an oligonucleotide ligated to pBR322, whose sequence was present in the 5' leader, to prime the synthesis of the second strand. With this method the percentage of recombinants containing E4 sequences ranged between 15 and 50% of the total colonies. Virtually all of these E4 cDNA clones contained the 5' leader sequence and several hundred were analyzed by comparing the results from single channel dideoxy sequencing reactions. Nine unique sequence patterns were identified and representative clones were completely sequenced.

Adenoviruses, Human↗

PubMed1985

Messenger RNAs from the transforming region of bovine papilloma virus type I.

Messenger RNAs present in C127 mouse cells transformed by bovine papilloma virus type 1 (BPV-1) were studied by the S1 nuclease protection technique, Northern blotting, and electron microscopic heteroduplex analysis. The results revealed at least five classes of spliced mRNAs which we designate types 1 to 5. They had a common poly(A) addition site located at co-ordinate 53 and all mRNAs, except the type 3 mRNAs, contained an exon located between co-ordinates 41 and 53. In the type 1 mRNAs this exon was connected to a very short leader sequence located around co-ordinate 31. The type 2 mRNAs contained 220 to 400-nucleotide long leaders which were located approximately 1.5 X 10(3) base-pairs further upstream. Two different subclasses of type 2 molecules (2A and 2B) were identified and these had slightly different leaders. The type 4 mRNAs contained a bipartite leader, whereas the type 5 mRNAs carried an approximately 900-nucleotide long leader. The type 3 mRNAs consisted of a main exon located between co-ordinates 32 and 53, linked to the same leader as is present in the type 2A mRNAs. A cap site which presumably is utilized by the type 2A, type 3, type 4 and type 5 mRNAs was mapped at nucleotide 89 in the BPV-1 sequence. A putative cap site for the type 1 mRNAs was mapped at co-ordinate 31.

Animals↗

PubMed1999

Gender-specific gene expression in Brugia malayi.

Brugia malayi is a mosquito-borne filarial nematode that causes lymphatic filariasis and elephantiasis in humans. The purpose of this study was to identify and characterize genes that are expressed differentially in male and female B. malayi in hopes of gaining new insight into the reproductive biology of the parasite. Two approaches were used. A 5' differential display PCR (splice leader differential display PCR, SL DD-PCR) was performed by PCR with splice leader and random primers on cDNA templates, and electronic subtraction was performed on expressed sequence tag (EST) cluster databases developed by the Filarial Genome Project (FGP). Gender-specific expression of candidate clones was confirmed by RT-PCR for six of 22 (27%) clones identified by DD and in seven of 15 (47%) clones identified by electronic subtraction. One clone was identified by both methods. Several female-specific clones had homology to known nematode genes that encode a fatty acid binding protein, a high mobility group protein, an eggshell protein, a glutamate-gated ion channel, and a collagen. However, most of the clones have no significant homology to known genes or proteins in computer databases. This project has confirmed the value of SL DD-PCR and electronic subtraction for analysis of gene expression in filariae. These two complimentary techniques may be generally applicable to the study of gender-specific (and by analogy stage specific) gene expression in other nematodes.

Animals↗

PubMed1986

Processing of mammalian tRNA transcripts in vitro: different pre-tRNAs are processed along alternative pathways that contain a common rate-limiting step.

We have analyzed the pathways and kinetics of processing of mouse tRNA gene transcripts in vitro. Different transcripts are processed along two alternative pathways. The 3' trailer sequence of the tRNA His primary transcript is excised before the 5' leader sequence. In contrast, for the tRNA Gly primary transcript, the 5' leader sequence is excised before the 3' trailer sequence, as has been found for other monomeric eukaryotic tRNA gene transcripts. Computerized analysis of the kinetics of processing indicates that tRNA Asp, tRNA Gly, tRNA Glu and tRNA His transcripts are processed in a substrate concentration-dependent manner and also reveals the existence of a common rate-limiting step, the rate constant of which is equivalent for three of the four transcripts tested. The processing of one pre-tRNA transcript can be competitively inhibited by addition of another pre-tRNA transcript to the processing reaction. The common rate-limiting step is associated with the conversion of the primary transcript to an intermediate and is independent of sequence and the particular processing pathway of the transcript.

Animals↗

PubMed1995

Most mRNAs in the nematode Ascaris lumbricoides are trans-spliced: a role for spliced leader addition in translational efficiency.

Some pre-mRNAs in nematodes are processed by trans-splicing. In this reaction, a 22-nt 5' terminal exon (the spliced leader, SL) and its associated 2,2,7-trimethylguanosine cap are acquired from a specialized Sm snRNP, the SL RNP. Although it has been evident for many years that not all nematode mRNAs contain the SL sequence, the prevalence of trans-spliced mRNAs has, with the exception of Caenorhabditis elegans, not been determined. To address this question in an organism amenable to biochemical analysis, we have prepared a message-dependent protein synthesis system from developing embryos of the parasitic nematode, Ascaris lumbricoides. Using this system, we have used both hybrid-arrest and hybrid-selection approaches to show that the vast majority (80-90%) of A. lumbricoides mRNAs contain the SL sequence and therefore are processed by trans-splicing. Furthermore, to examine the effect of SL addition on translation, we have measured levels of protein synthesis in extracts programmed with a variety of synthetic mRNAs. We find that the SL sequence itself and its associated hypermethylated cap functionally collaborate to enhance translational efficiency, presumably at the level of initiation of protein synthesis. These results indicate that trans-splicing plays a larger role in nematode gene expression than previously suspected.

Animals↗

PubMed1994

Properties of variant forms of human stem cell factor recombinantly expressed in Escherichia coli.

The gene for human stem cell factor (SCF) encodes a leader sequence followed by 248 amino acids (Martin et al., 1990, Cell 63, 203). Of these 248 amino acids, the first 189 correspond to an extracellular domain and the remainder correspond to a hydrophobic transmembrane domain plus a cytoplasmic domain. A naturally occurring soluble form, released by proteolytic cleavage after amino acid 165, has been described. An alternatively spliced mRNA, lacking the codons for exon 6, has also been described. Since the amino acids encoded by exon 6 include the proteolytic cleavage site, the form expressed from the alternatively spliced mRNA tends to remain membrane-bound. In the present study, we have begun to explore structure/function relationships within the extracellular domain of SCF. Forms beginning at amino acid 1 (after the leader sequence) and ranging from 127 to 189 at the C-terminus have been recombinantly expressed in Escherichia coli and purified. In addition, forms missing the amino acids encoded by exon 6, forms missing up to 10 amino acids from the N-terminus, and forms with disulfide bond alterations have been expressed and purified. The forms have been characterized structurally, as well as functionally, in quantitative cell proliferation and receptor-binding assays. The results indicate that amino acids 1-141 comprise a structural and functional core and allow conclusions about the necessity of each of the two disulfide bonds for structure and function.

Alternative Splicing↗

PubMed1992

Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens.

We have investigated HLA-G mRNA expression in cells and tissues expressing the gene. This analysis has demonstrated that the HLA-G primary transcript is alternatively spliced to yield at least three distinct mature mRNAs. Sequencing of the transcripts has shown that the largest mRNA is essentially that previously characterized, encoding a leader sequence, three external domains, a transmembrane region, and a cytoplasmic sequence. Of the two smaller messages, a 900-base mRNA does not include exon 3, resulting in a predicted protein sequence with the alpha 1 and alpha 3 external domains joined. The smallest mRNA results from splicing out exons 3 and 4, connecting the alpha domain directly to the transmembrane sequence. Alternative splicing of HLA-G mRNA was found in placental tissues and in eye tissue as well as in HLA-G-transfected cell lines. In term placental tissue the smallest mRNA appeared to be more abundant than the full-length form, while in a cell line derived from an earlier developmental stage the larger form predominated. Immunoprecipitation of [35S]methionine-labeled cell lysates showed that three different HLA-G proteins were present in transfected cells, with sizes corresponding to those predicted from the three alternative mRNA sequences. These findings are discussed in terms of potential functions of the alternative HLA-G proteins.

Amino Acid Sequence↗