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PubMed1992

Domain structure of U2 and U4/U6 small nuclear ribonucleoprotein particles from Trypanosoma brucei: identification of trans-spliceosomal specific RNA-protein interactions.

Maturation of mRNAs in trypanosomes involves trans splicing of the 5' end of the spliced leader RNA and the exons of polycistronic pre-mRNAs, requiring small nuclear ribonucleoproteins (snRNPs) as cofactors. We have mapped protein-binding sites in the U2 and U4/U6 snRNPs by a combination of RNase H protection analysis, native gel electrophoresis, and CsCl density gradient centrifugation. In the U2 snRNP, protein binding occurs primarily in the 3'-terminal domain; through U2 snRNP reconstitution and chemical modification-interference assays, we have identified discrete positions within stem-loop IV of Trypanosoma brucei U2 RNA that are essential for protein binding; significantly, some of these positions differ from the consensus sequence derived from cis-spliceosomal U2 RNAs. In the U4/U6 snRNP, the major protein-binding region is contained within the 3'-terminal half of U4 RNA. In sum, while the overall domain structure of the U2 and U4/U6 snRNPs is conserved between cis- and trans-splicing systems, our data suggest that there are also trans-spliceosomal specific determinants of RNA-protein binding.

Animals↗
PubMed1995

Leishmania RNA virus 1-mediated cap-independent translation.

Recently, a group of related Leishmania RNA viruses (Leishmania RNA virus 1 [LRV1]) has been isolated from Leishmania guyanensis and L. brasiliensis. These viruses persist in the cytoplasm and contain double-stranded RNA genomes. Miniexon sequences are absent from the 5' end of the viral RNA, and the 5' end of the viral RNA lacks a cap structure, suggesting that LRV1 has evolved a cap-independent mechanism of translation. Cap-independent translation of picornavirus genomic RNA requires a cis element, within the 5' untranslated region (UTR), referred to as an internal ribosome entry site (IRES). In order to find out if the 5' UTR of LRV1 possessed IRES activity, we modified a Leishmania expression vector, pX63NEO-GUS, so that it would produce a dicistronic transcript in which the neomycin phosphotransferase gene was separated from the downstream beta-glucuronidase (GUS) gene by the LRV1 5' UTR. High levels of GUS activity were detected in L. major stably transformed with this plasmid. Elimination of the first 120 nucleotides of the viral 5' UTR lowered GUS activity 10-fold. Furthermore, when the entire 5' UTR was eliminated, GUS activity was undetectable. These results, together with the absence of trans-spliced GUS transcripts, are consistent with the hypothesis that the 5' UTR of LRV1 functions as an IRES element. The ability to couple expression of genes via an IRES element should prove useful in genetic experiments with Leishmania spp.

Animals↗
PubMed1988

The cap of both miniexon-derived RNA and mRNA of trypanosomes is 7-methylguanosine.

Most, if not all, trypanosome mRNAs have the same 35-base sequence at their 5' terminus which is derived from a short RNA (medRNA) probably by the process of trans-splicing. It is of interest, evolutionarily and mechanistically, to determine the chemical structure of the 5' terminus of the precursor (medRNA) and product (mRNA). We demonstrate here that the cap structure of both is most probably 7-methylguanosine in a 5',5' triphosphate linkage, consistent with a precursor/product relationship.

Animals↗
PubMed1988

Regulatory myosin light-chain genes of Caenorhabditis elegans.

We have cloned and analyzed the Caenorhabditis elegans regulatory myosin light-chain genes. C. elegans contains two such genes, which we have designated mlc-1 and mlc-2. The two genes are separated by 2.6 kilobases and are divergently transcribed. We determined the complete nucleotide sequences of both mlc-1 and mlc-2. A single, conservative amino acid substitution distinguishes the sequences of the two proteins. The C. elegans proteins are strongly homologous to regulatory myosin light chains of Drosophila melanogaster and vertebrates and weakly homologous to a superfamily of eucaryotic calcium-binding proteins. Both mlc-1 and mlc-2 encode abundant mRNAs. We mapped the 5' termini of these transcripts by using primer extension sequencing of mRNA templates. mlc-1 mRNAs initiate within conserved hexanucleotides at two different positions, located at -28 and -38 relative to the start of translation. The 5' terminus of mlc-2 mRNA is not encoded in the 4.8-kilobase genomic region upstream of mlc-2. Rather, mlc-2 mRNA contains at its 5' end a short, untranslated leader sequence that is identical to the trans-spliced leader sequence of three C. elegans actin genes.

Amino Acid Sequence↗
PubMed1989

UbiA, the major polyubiquitin locus in Caenorhabditis elegans, has unusual structural features and is constitutively expressed.

Ubiquitin is a multifunctional 76-amino-acid protein which plays critical roles in many aspects of cellular metabolism. In Caenorhabditis elegans, the major source of ubiquitin RNA is the polyubiquitin locus, UbiA. UbiA is transcribed as a polycistronic mRNA which contains 11 tandem repeats of ubiquitin sequence and possesses a 2-amino-acid carboxy-terminal extension on the final repeat. The UbiA locus possesses several unusual features not seen in the ubiquitin genes of other organisms studied to date. Mature UbiA mRNA acquires a 22-nucleotide leader sequence via a trans-splicing reaction involving a 100-nucleotide splice leader RNA derived from a different chromosome. UbiA is also unique among known polyubiquitin genes in containing four cis-spliced introns within its coding sequence. Thus, UbiA is one of a small class of genes found in higher eucaryotes whose heterogeneous nuclear RNA undergoes both cis and trans splicing. The putative promoter region of UbiA contains a number of potential regulatory elements: (i) a cytosine-rich block, (ii) two sequences resembling the heat shock regulatory element, and (iii) a palindromic sequence with homology to the DNA-binding site of the mammalian steroid hormone receptor. The expression of the UbiA gene has been studied under various heat shock conditions and has been monitored during larval moulting and throughout the major stages of development. These studies indicate that the expression of the UbiA gene is not inducible by acute or chronic heat shock and does not appear to be under nutritional or developmental regulation in C. elegans.

Animals↗
PubMed1989

The conserved U.G pair in the 5' splice site duplex of a group I intron is required in the first but not the second step of self-splicing.

Group I self-splicing introns have a 5' splice site duplex (P1) that contains a single conserved base pair (U.G). The U is the last nucleotide of the 5' exon, and the G is part of the internal guide sequence within the intron. Using site-specific mutagenesis and analysis of the rate and accuracy of splicing of the Tetrahymena thermophila group I intron, we found that both the U and the G of the U.G pair are important for the first step of self-splicing (attack of GTP at the 5' splice site). Mutation of the U to a purine activated cryptic 5' splice sites in which a U.G pair was restored; this result emphasizes the preference for a U.G at the splice site. Nevertheless, some splicing persisted at the normal site after introduction of a purine, suggesting that position within the P1 helix is another determinant of 5' splice site choice. When the U was changed to a C, the accuracy of splicing was not affected, but the Km for GTP was increased by a factor of 15 and the catalytic rate constant was decreased by a factor of 7. Substitution of U.A, U.U, G.G, or A.G for the conserved U.G decreased the rate of splicing by an even greater amount. In contrast, mutation of the conserved G enhanced the second step of splicing, as evidenced by a trans-splicing assay. Furthermore, a free 5' exon ending in A or C instead of the conserved U underwent efficient ligation. Thus, unlike the remainder of the P1 helix, which functions in both the first and second steps of self-splicing, the conserved U.G appears to be important only for the first step.

Animals↗
PubMed1989

Molecular biology of Leishmania.

Leishmania is a trypanosomatid protozoa with a digenetic life cycle. Sandflies inject promastigotes, the free living form present in their salivary glands, into mammals where the parasite colonizes macrophages, transforming into intracellular amastigotes. The cycle is completed when during a blood meal the insect ingests infected macrophages, the amastigotes are released in the gut where they transform back into promastigotes. Leishmania has to adapt to the changing life conditions, from free-living forms in the poikilothermic insect vector to obligatory intracellular parasite in the homeothermic mammalian host. It also has to adapt to the acidic pH of the macrophage's phagolysosome where amastigotes multiply. The adaptative response of Leishmania includes morphological, physiological, and biochemical changes. Promastigotes can be grown in culture medium. Studies of changes taking place during adaptation have been facilitated by the establishment of in vitro conditions that allow the transformation of amastigotes into promastigotes and vice versa. The system is well suited for studying regulation of gene expression during adaptative differentiation. Some mechanisms of mRNA processing are unique to these protozoa: trans-splicing and RNA editing. Several genes that are differentially expressed in the two stages have been studied. No obvious cis regulatory motifs have been found in the DNA.

Amino Acid Sequence↗
PubMed1986

Discontinuous transcription and antigenic variation in trypanosomes.

The main theme of this review is the discontinuous synthesis of mRNAs in trypanosomes. This novel process was discovered in the unicellular eukaryote Trypanosoma brucei, but it is probably a general feature of the order of Kinetoplastida, to which several other major human pathogens belong. Discontinuous RNA synthesis involves a sequence of 35 nucleotides (nt) found at the 5' end of all trypanosome mRNAs analyzed. The 35-nt sequence is encoded in arrays of 1.35-kb repeats that are clustered in the genome. The primary transcript of the 1.35-kb repeat is an RNA of 140 nt that carries the 35-nt sequence at its 5' end. The 35-nt sequence is transferred from the 140-nt precursor to pre-mRNAs made elsewhere in the genome. The process has not yet been reconstructed in vitro, and whether transfer involves priming of pre-mRNA synthesis, RNA-RNA ligation followed by splicing, trans-splicing, or more than one of these mechanisms, is still unknown. Circumstantial evidence makes priming the least likely of these alternatives. Why it is advantageous to trypanosomes to make their mRNAs in such an unusual fashion is unclear. As yet, there is no evidence for discontinuous synthesis of mRNAs in organisms other than kinetoplastid flagellates. The mini-exon sequence was first found in mRNAs for Variant-specific Surface Glycoproteins (VSGs), and the control of the synthesis of these proteins is a second theme of this review. Silent VSG genes may be activated by their duplicative transposition to a telomeric expression site. The transposition process looks like a gene conversion, mediated by short blocks of sequence homology. Activation of the transposed gene is due to its insertion into an active transcription unit, i.e. to promoter addition. Telomeric VSG genes can also be activated without duplication. This can occur by a reciprocal translocation in which a silent telomeric gene exchanges position with a gene residing in an active expression site. A VSG gene may also be activated without detectable translocation, however, by the transcriptional activation of the silent expression site in which it is located. How this occurs is still unknown, because the transcription units are so long that the promoter for pre-mRNA synthesis has not yet been reached by chromosome walking. A simple mechanism in which a mobile promoter moves between telomeres has been rendered unlikely by the demonstration that two telomeric transcription units can be simultaneously active when one of them is interrupted by a large DNA insertion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Molecular mechanisms and therapeutic approaches to the treatment of African trypanosomiasis.

There are only a handful of drugs available today for treating African trypanosomiasis, most of which were discovered more than forty years ago. These drugs are plagued by various problems, ranging from oral in-absorption, acute toxicities, short durations of action, and low efficacies to the emergence of trypanosomal resistance. Mechanisms of antitrypanosomal action of these drugs are mostly unknown, except for eflornithine, which is a suicide inhibitor of ornithine decarboxylase. On the other hand, the African trypanosomes are among the most extensively studied parasitic protozoa to date. Many of their intriguing biological features have been well documented and can be viewed as attractive targets for antitrypanosomal chemotherapy. These features include the glycosomal functions and protein import, the trans-splicing of mRNAs, the machineries for controlled protein degradations, the polyamine metabolism, the trypanothione metabolism, the purine salvage enzymes, and the glycolipid anchor for the surface glycoproteins.

Animals↗
PubMed2025

Proteomics-based evaluation of AAV dystrophin gene therapy outcomes in mdx skeletal muscle.

Duchenne muscular dystrophy (DMD) is a fatal genetic muscle-wasting disease characterized by loss of dystrophin protein. Therapeutic attempts to restore a functional copy of dystrophin to striated muscle are under active development, and many utilize adeno-associated viral (AAV) vectors. However, the limited cargo capacity of AAVs precludes delivery of full-length dystrophin, a 427 kDa protein, to target tissues. Recently, we developed a method to express large dystrophin constructs using the protein trans-splicing mechanism mediated by split inteins and myotropic AAV vectors. The efficacy of this approach to restore muscle function in mdx4cv mice was previously assessed using histology, dystrophin immunolabeling, and Western blotting. Here, we expand our molecular characterization of dystrophin constructs with variable lengths using a mass spectrometry-based proteomics approach, providing insight into unique protein expression profiles in skeletal muscles of wild-type, dystrophic mdx4cv, and AAV-treated mdx4cv mice. Our data reveal several affected cellular processes in mdx4cv skeletal muscles with changes in the expression profiles of key proteins to muscle homeostasis, whereas successful expression of dystrophin constructs results in an intermediate to complete restoration. This study highlights several biomarkers that could be used in future preclinical or clinical studies to evaluate the effectiveness of therapeutic strategies.

Animals↗
PubMed1992

[Advances in molecular parasitology].

Recent studies on molecular biology of parasites revealed novel mechanisms for the control of expression of parasitic genes. In trypanosomatid protozoa, unusual characteristics of gene regulation, including polycistronic transcription, trans-RNA splicing and RNA editing have been discovered. This review describes the recent progress of RNA editing in trypanosomes, and trans-splicing in both trypanosomes and nematodes. Studies of the editing of mRNAs in trypanosomatid mitochondria indicate the involvement of small guide RNA molecules, which are encoded in the minicircles and/or maxicircles of kinetoplast DNA. A proposed transesterification model for RNA editing suggests an analogous reaction between RNA editing and RNA splicing. In addition, the development of stable transfection system and success of homologous gene targeting in Trypanosoma and Leishmania are reported.

Animals↗
PubMed1991

Casein kinase II from Caenorhabditis elegans. Cloning, characterization, and developmental regulation of the gene encoding the beta subunit.

Complementary DNAs encoding the beta subunit of casein kinase II (CKII beta) from the nematode Caenorhabditis elegans were cloned and sequenced. The predicted beta subunit polypeptide comprises 234 amino acid residues and has a Mr of 26,452. CKII beta is not homologous with other types of proteins. In synchronously developing C. elegans the abundance of the 1.3-kilobase mRNA for CKII beta varies in parallel with the level of mRNA encoding the catalytic subunit (alpha) of CKII. Thus, the developmental expression of CKII subunits is controlled coordinately and pretranslationally. CKII beta and CKII alpha mRNAs are enriched 5-10-fold in C. elegans embryos relative to their concentrations at several other stages of nematode development. A 3.8-kilobase pair segment of C. elegans DNA that contains the CKII beta gene and an extensive 5'-flanking region was cloned and sequenced. The CKII beta gene is divided into 6 exons by introns ranging from 49 to 533 base pairs in length. The first exon encodes 88 nucleotides of 5'-untranslated mRNA. Exon 2 (72 base pairs) contains the initiator Met codon and only 5 additional codons. Exons 3-6 encode 52, 63, 64, and 49 amino acid residues, respectively. The 5' terminus of CKII beta mRNA is modified post-transcriptionally by trans-splicing with a leader sequence of 22 nucleotides. The CKII beta gene was mapped to a position on C. elegans chromosome 2 that is in close proximity to the lin-11 gene.

Amino Acid Sequence↗
PubMed1990

Cloning, characterization, and expression of the gene for the catalytic subunit of cAMP-dependent protein kinase in Caenorhabditis elegans. Identification of highly conserved and unique isoforms generated by alternative splicing.

The nematode Caenorhabditis elegans expresses substantial amounts of several forms (Mr values = 39,000-41,000) of the catalytic subunit (C) of cAMP-dependent protein kinase. Approximately 65% of the total cAMP-dependent phosphotransferase activity is recovered in particulate fractions of homogenates prepared from asynchronous populations of C. elegans. The C subunit is expressed at a low level in cytosolic and particulate compartments during embryogenesis. As the nematodes progress from late embryonic stages to the newly hatched, first larval (L1) stage, C subunit content increases 15-fold. High levels of C subunits are observed in several subsequent larval and adult stages of development. Since the relative abundance of C subunit mRNA changes little with development, it appears that control of C expression is exerted the translational and/or post-translational levels. cDNAs for two types of C have been cloned and sequenced. The derived amino acid sequence of a major isoform (CeCAT alpha, 358 residues) is highly homologous (82% identical) with the murine C alpha subunit. A second, novel C subunit (CeCAT alpha', 374 residues) has a unique 56-residue carboxyl-terminal region that is generated by the alternative splicing of the C pre-mRNA. The splicing process that yields CeCAT alpha' is unusual because it converts the central portion of an apparent 1-kilobase (kb) intron to an exon. The alternative exon introduces the novel carboxyl terminus and a new translation stop signal, while simultaneously converting the coding sequence for 40 carboxyl-terminal residues in CeCAT alpha into 3'-untranslated nucleotides. The 5' end of the C. elegans C subunit mRNA is produced by the trans-splicing of the C gene transcript to a 22-base pair C. elegans leader sequence originally described by Krause, M., and Hirsh, D. [1987) Cell 49, 753-761). The 20-kb C. elegans C gene is divided into seven exons by introns ranging in size from 54 to 8000 bp. The sizes of the C. elegans C subunit gene, cytoplasmic mRNA (2.5 kb), and subunit protein are similar to the sizes of the murine C alpha gene, mRNA, and polypeptide. However, the nematode and murine C genes differ significantly in the organization of their introns and exons.

Amino Acid Sequence↗
PubMed1989

Chloroplast ribosomal protein gene rps12 of Chlamydomonas reinhardtii. Wild-type sequence, mutation to streptomycin resistance and dependence, and function in Escherichia coli.

The chloroplast rps12 gene encoding ribosomal protein S12 from wild-type as well as from streptomycin-resistant and -dependent mutants of Chlamydomonas reinhardtii was cloned and sequenced. At least six mRNA transcripts ranging in size from 800 to 4800 nucleotides were found to contain the rps12 coding sequence. As in Escherichia coli and Euglena, the C. reinhardtii rps12 gene is continuous, in contrast to its trans-spliced structure in higher plants. However, rps12 of C. reinhardtii is no longer immediately adjacent to the rps7 gene encoding ribosomal protein S7 as it is in other organisms. The deduced amino acid sequence of C. reinhardtii S12 protein shows strong homologies (48-79% identity) to S12 protein sequences of other organisms, but it has extra amino acid residues at its C terminus. Single base pair changes at different sites result in streptomycin-resistant or -dependent mutants with amino acid changes identical to comparable mutations in the E. coli S12 protein. The chloroplast rps12 gene is also expressed under the control of its own promoter in E. coli cells, and the C. reinhardtii S12 protein produced assembles into the E. coli ribosomes where it appears to function efficiently.

Amino Acid Sequence↗
PubMed1989

Molecular characterization of the Trypanosoma brucei RNA polymerase I and III largest subunit genes.

We have sequenced a 7.1-kilobase fragment of the Trypanosoma brucei RNA polymerase (pol) I largest subunit gene. The 6.9-kilobase transcript from this gene is of roughly equal abundance in bloodstream- and procyclic-form trypanosomes and has a 39-nucleotide trans-spliced leader 290 nucleotides upsteam of the putative initiation codon. The 1781-amino acid trypanosome pol I polypeptide is considerably less similar to its yeast homolog than the trypanosome pol II and pol III polypeptides are to their counterparts in yeast. The yeast and trypanosome pol I polypeptides are distinguished from pol II and pol III largest subunits by two inserts, 100-200 amino acids in length, which are found in the nonconserved portions of the polypeptides. We also report our sequence of the trypanosome pol III largest subunit gene, which agrees with that reported by Köck et al. (Köck, J., Evers, R., and Cornelissen, A.W.C.A. (1988) Nucleic Acids Res. 16, 8753-8772) except for 13 nucleotide differences. Four spliced leader addition sites between 60 and 99 nucleotides upstream of the AUG initiation codon were observed for the 5.4-kilobase transcript from this gene. Four short stretches of amino acid homology between the yeast and trypanosome pol III largest subunit polypeptides were identified which appear to be specific to this class of RNA polymerase; only one short pol I-specific sequence was identified in our comparison of the yeast and trypanosome pol I largest subunit polypeptides.

Amino Acid Sequence↗
PubMed1995

In vivo structural analysis of spliced leader RNAs in Trypanosoma brucei and Leptomonas collosoma: a flexible structure that is independent of cap4 methylations.

The formation of the mRNA 5' end in trypanosomatid protozoa is carried out by trans-splicing, which transfers a spliced leader (SL) sequence and its hypermethylated cap (cap4) from the SL RNA to the pre-mRNA. Previous in vitro studies with synthetic uncapped RNAs have shown that the SL sequence of Leptomonas collosoma can assume two alternate conformations, Form 1 and Form 2, with Form 1 being the dominant one. To gain information about the structure of the SL RNA in vivo, in its protein-rich environment, we have used permeable Trypanosoma brucei and L. collosoma cells for chemical modification experiments. We introduce the use in vivo of the water-soluble reagents CMCT and kethoxal. In contrast to the in vitro results, the Form 2 secondary structure predominates. However, there are chemically accessible regions that suggest conformational flexibility in SL RNPs and a chemically inaccessible region suggestive of protection by protein or involvement in tertiary interactions. Using complementary 2'-O-methyl RNA oligonucleotides, we show that T. brucei SL RNA can be induced to switch conformation in vivo. SL RNA stripped of proteins and probed in vitro does not display the same Form 2 bias, indicating that SL RNA structure is determined, at least in part, by its RNP context. Finally, the methyl groups of the cap4 do not seem to affect the secondary structure of T. brucei SL RNA, as shown by chemical modification of undermethylated SL RNA probed in vivo.

Aldehydes↗
PubMed1995

Germ-line human epsilon heavy chain gene RNA transcripts utilize the full range of alternative 3' splicing seen in productive epsilon mRNA.

An early step in immunoglobulin isotype switching involves the initiation of active transcription of downstream heavy chain loci while they are still in germ-line configuration. This results in the production of 'sterile' germ-line RNA transcripts that do not contain VDJ sequences, the role of which in isotype switching is under intense scrutiny. We investigated whether such human epsilon germ-line transcripts employ the full complement of complex alternative 3' RNA splicing, splicing we have recently reported occurring with productive epsilon mRNA transcripts. Using a reverse transcriptase polymerase chain reaction (RT-PCR) strategy, in which the 5' primer was located in the I region of the epsilon gene, a region expressed in germ-line but not productive (VDJ containing) epsilon transcripts, we showed that the full range of alternative 3' epsilon splices occur with germ-line transcripts. These results demonstrate that epsilon 3' splicing events are independent of 5' isotype DNA switching. Additionally, we showed that, just as with mature epsilon mRNA, the relative production of the various epsilon germ-line mRNA isoforms was responsive to modulation by stimuli such as interleukin-10 (IL-10). Thus B cells, when stimulated to produce epsilon germ-line transcripts, generate a family of germ-line mRNA that differ not only in their initiation sites but, more importantly, also differ in their 3' sequences, sequences that could be important in regulation of the parent gene itself. Furthermore, by discontinuous or trans-splicing, cells could utilize these various epsilon germ-line transcripts to produce the full range of mature IgE proteins prior to undergoing deletional recombination of isotype switching.

Base Sequence↗
PubMed1993

Characterization of cDNA clones for the 2-methyl branched-chain enoyl-CoA reductase. An enzyme involved in branched-chain fatty acid synthesis in anaerobic mitochondria of the parasitic nematode Ascaris suum.

The 2-methyl branched-chain enoyl-CoA reductase plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode Ascaris suum. An affinity-purified polyclonal anti-serum against the reductase was used to screen a cDNA library constructed in lambda gt11 with poly(A)+ RNA from adult A. suum muscle. A 1.2-kilobase partial cDNA clone was isolated, subcloned, and sequenced in both directions. Additional sequence at the 5' end of the mRNA was determined by the RACE (rapid amplification of cDNA ends) procedure. Nucleotide sequence analysis of the cDNAs revealed the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1236 nucleotides and a 3'-untranslated sequence of 109 nucleotides including a short poly(A) tail 14 nucleotides from a polyadenylation signal (AATAAA). The open reading frame encoded a 396-amino acid sequence (M(r) 43,046) including a 16-amino acid leader peptide. Two-dimensional gel electrophoresis of the purified reductase yielded multiple spots with two distinct but overlapping amino-terminal amino acid sequences. Both sequences overlapped with the sequence predicted from the mRNA, and one of the sequences was identical to the predicted sequence. Comparison of the ascarid sequence with that of mammalian acyl-CoA dehydrogenases revealed a high degree of sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene even though the ascarid enzyme functions as a reductase, not as a dehydrogenase. Immunoblotting of A. suum larval stages and adult tissues with antisera that cross-reacted with each of the spots separated on two-dimensional gels suggested that the reductase was only found in adult muscle. Northern blotting using the partial cDNA revealed a hybridization band of about 1.5 kilobases and also suggested that the enzyme was tissue-specific and developmentally regulated in agreement with the results of the immunoblotting.

Amino Acid Sequence↗