Characterization of two distinct gene transcripts for ribosomal protein L21 from pathogenic and nonpathogenic strains of Entamoeba histolytica.

A second gene (rp-L21) copy, clone g34, coding for ribosomal (r-) protein L21, was isolated from the pathogenic (P) strain HM-1:IMSS cl6 of the intestinal parasite Entamoeba histolytica (Eh). The gene was compared to the previously isolated copy, gLE3 [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], with respect to its primary structure, mRNA levels and binding to the r-complex during translation. Unlike the gLE3 gene copy [Petter et al., Mol. Biochem. Parasitol. 56 (1992) 329-334], g34 was found not to be physically connected to an actin gene copy. Homologous copies of the two rp-L21 genes were also characterized from the nonpathogenic (NP) strain SAW1734R clAR, as well as from its P derivative. Sequence comparison of the coding regions of the two rp-L21 revealed almost full identity. Significant differences were found, however, within their 3' and 5' flanking regions. Using the 3' rapid amplification of cDNA ends (3' RACE) method [Frohman et al., Proc. Natl. Acad. Sci. USA 85 (1988) 8998-9002], as well as Northern and slot blot hybridizations, it was demonstrated that both rp-L21 mRNAs are found in similar amounts. However, as was shown by differential hybridization, the relative binding of each transcript to the r-complex varied somewhat between P and NP strains. This finding suggests that the control of expression of rp-L21 in Eh may involve regulation at the post-transcriptional level.

Cloning and sequence analysis of the Erythrina corallodendron lectin cDNA.

Examination of the hemagglutinating activity of extracts from seeds of Erythrina corallodendron at various maturation stages revealed that the level of lectin increases markedly past mid-maturation. Seeds at this stage of maturation served as a source of mRNA for the construction of an expression cDNA library in the vector lambda Zap, which generates fusion proteins with an N-terminal portion of beta-galactosidase. The library was screened with rabbit polyclonal anti-ECorL antiserum. Four immunopositive clones were isolated. Western blot analysis of cell extracts from one of the clones (pIEcL-B) showed a 36 kDa protein that reacted with the antiserum, as well as with a mouse monoclonal antibody raised against the lectin. DNA sequence analysis by the chain termination method revealed that clone pIEcl-C has an insert of 1017 bp with the entire coding sequence of ECorL, beginning with an initiation codon ATG at position 26 and ending with stop codon TAA at position 868. This fragment encodes a polypeptide of 281 amino acids consisting of a signal leader sequence of 25 amino acids and a mature protein of 256 amino acids. The deduced amino acid sequence from this fragment is identical to the sequence of the first 244 amino acids of ECorL, as determined at the protein level, except at 7 positions.

Modification by site-directed mutagenesis of the specificity of Erythrina corallodendron lectin for galactose derivatives with bulky substituents at C-2.

Examination of the three-dimensional structure of Erythrina corallodendron lectin (ECorL) in complex with a ligand (lactose), the first of its kind for a Gal/GalNAc-specific lectin [(1991) Science 254, 862-866], revealed the presence of a hydrophobic cavity, surrounded by Tyr108 and Pro134-Trp135, which can accommodate bulky substituents such as acetamido or dansylamido (NDns) at C-2 of the lectin-bound galactose. Comparison of the primary sequence of ECorL with that of soybean agglutinin, specific for galactose and its C-2 substituted derivatives, and of peanut agglutinin, specific for galactose only, showed that in soybean agglutinin, Tyr108 is retained, and Pro134-Trp135 is replaced by Ser-Trp, whereas in peanut agglutinin, the former residue is replaced by Thr and the dipeptide by Ser-Glu- Tyr-Asn. Three mutants of ECorL were therefore constructed: L2, in which Pro134-Trp135 was replaced by Ser-Glu-Tyr-Asn; Y108T, in which Tyr108 was replaced by Thr and the double mutant L2; Y108T. They were expressed in Escherichia coli, as done for recombinant ECorL [(1992) Eur. J. Biochem. 205, 575-581]. The mutants had the same hemagglutinating activity as native or rECorL. Their specificity for galactose, GalNAc and Me beta GalNDns was examined by inhibition of hemagglutination and of the binding of the lectin to immobilized asialofetuin; in addition, their association constants with Me alpha GalNDns and Me beta GalNDns were measured by spectrofluorimetric titration. The results showed that Y108T had essentially similar specificity as the native and recombinant lectins. The affinity of L2 and L2;Y108T for galactose was also the same as ECorL, but they had a lower affinity for GalNAc and markedly diminished affinity for the dansyl sugars (up to 43 times, or 2 kcal, less). This appears to be largely due to steric hindrance by the two additional amino acids present in the cavity region in these mutants. Our findings also provide an explanation for the inability of PNA to accommodate C-2-substituted galactose derivatives at its primary subsite.

Cloning and characterization of an unusual elongation factor-1 alpha cDNA from Entamoeba histolytica.

The coding sequence deduced from two overlapping cDNA inserts obtained from a pathogenic strain of Entamoeba histolytica revealed a striking homology (greater than 85%) with elongation factor EF-1 alpha from Saccharomyces cerevisiae and Artemia salina. The deduced amino acid sequence predicted a size of 49 kDa, and antibodies raised against the S. cerevisiae EF-1 alpha cross-reacted with an amoebic protein of similar size (45-47 kDa). Sequence analysis of the cDNA revealed that the 5' untranslated region contained a stretch of 190 nucleotides which was perfectly complementary to a segment of the 3' terminal coding region situated 1015 bases downstream of the methionine initiation codon. Electron microscopy of self-renatured cDNA confirmed the potential of such molecules to form a stem-loop secondary structure. The presence of the complementary sequences was confirmed at the genomic level by sequence analysis of polymerase chain reaction-amplified segments which span both the 3' and 5' terminal complementary regions. Comparison of the deduced amino acid sequence of E. histolytica EF-1 alpha with Ef-Tu from Escherichia coli and EF-1 alpha from different sources, suggested that the major functional domains of the protein are located within the loop structure.

Entamoeba histolytica: cloning and characterization of actin cDNA.

In order to study gene expression in the human parasite Entamoeba histolytica, a cDNA library of E. histolytica strain 200:NIH was constructed using the phage vector lambda gt10. Three cDNA clones (A, B and C) were selected for further analysis. Each of the three clones hybridized to a distinct mRNA. Two of these mRNAs were translated in vitro after hybrid selection, and yielded distinct translation products. One of these mRNAs, selected by hybridization to clone A, encodes the most abundantly expressed protein in E. histolytica. DNA sequence analysis of this cDNA clone identified the DNA as that encoding actin. The deduced amino acid sequence of E. histolytica actin resembles both cytoplasmic and muscle actins and has an unusual N-terminal glycine residue. We have shown that a family of actin genes is present in E. histolytica. Six different E. histolytica actin clones were obtained from a lambda gt10 genomic library using subcloned cDNA probes. Southern analysis of three different E. histolytica strains (200:NIH, Rhaman, and HM-1:IMSS) revealed at least four different actin genes. Strain HM-1:IMSS, however, differs by the presence of an additional actin gene.

Nucleotide sequence analysis of an Entamoeba histolytica ferredoxin gene.

A cDNA clone (subclone B) previously isolated from the human parasite Entamoeba histolytica was characterized. DNA sequence analysis of subclone B identified the DNA as that encoding apoferredoxin. E. histolytica ferredoxin cDNA contains unusually short 5' and 3' noncoding regions of 9 and 25 nucleotides, respectively. A genomic ferredoxin clone was isolated from E. histolytica DNA, and comparison of genomic and cDNA sequences revealed that the ferredoxin gene is unspliced. The deduced amino acid sequence of E. histolytica ferredoxin resembles clostridial type of ferredoxins, and shows an arrangement of cysteines characteristic for the coordination of 2[4Fe-4S] centres. Of interest is the absence of an aromatic amino acid in the N-terminal region of the protein, a feature which is conserved in clostridial ferredoxins. Southern blot analysis of three different E. histolytica strains (200:NIH, Rahman and HM-1:IMSS) demonstrated the presence of a family of at least two ferredoxin genes. One of these genes is marked by restriction length polymorphisms in different strains of E. histolytica.

Entamoeba histolytica ribosomal RNA genes are carried on palindromic circular DNA molecules.

Highly abundant DNA fragments obtained after restriction enzyme digests of nuclear DNA of Entamoeba histolytica strain HM-1:IMSS have been cloned and characterized. Northern blot hybridization to E. histolytica rRNA and sequence analysis identified the abundant DNAs as ribosomal DNA containing species. Several overlapping clones containing these abundant DNAs were isolated from 4 different genomic libraries of E. histolytica. Alignment of the restriction maps was consistent with a circular molecule, about 24.6 kilobase pairs (kb) in size. Nuclease BA131 digestion provided additional evidence for the circular nature of this DNA. The ribosomal DNA molecule contains two large inverted repeat-regions, each at least 5.2 kb in length. Sequence analysis of clone R715 revealed homology to the large rRNA units of various eukaryotic organisms. This clone was located in both inverted repeats, suggesting two rRNA cistrons per molecule. The inverted repeats are flanked by stretches of DNA which contain tandemly reiterated sequences. Southern blot analysis of E. histolytica nuclear DNA revealed the presence of two populations of molecules. These molecules have identical arrangements of restriction sites, but differ in size (0.7 kb) in a fragment containing tandemly reiterated sequences. Analysis of E. histolytica nuclear DNA by electron microscopy also revealed circular molecules. These molecules are about 26.6 kb +/- 0.5 kb in size and contain structural features predicted by the restriction map of the extrachromosomal ribosomal DNA of E. histolytica.

Chromosome walk in Entamoeba histolytica: the gene encoding for ribosomal protein L21 neighbors one of the actin genes.

Actin is one of the most abundant proteins in the motile intestinal protozoan parasite E. histolytica. A number of actin gene copies have been detected. The cDNA and genomic sequences of two of the actin genes have been independently reported (1,2). Almost complete homology was detected between the coding regions of the two genes; however, significant differences were observed in the sequences of their 5' untranslated regions. Using the coding region of actin as the focal point, we performed a chromosome walk to identify the neighboring genes and the intergenic regulatory domains. A genomic library containing large fragments of DNA was screened with the coding and non-coding regions of the actin gene. An insert of 8.5 kb reacted on Northern blots with actin and two additional transcripts. The large (approximately 2.5 kb) transcript has not yet been identified, but the smaller one (600 bp), was shown to encode for the ribosomal protein L21. Both the cDNA and genomic sequences of this gene were determined. The RP-L21 gene was found to be physically connected to the actin gene by a 2.1 kb intergenic stretch. The actin gene on this DNA fragment contained a 5' untranslated region that was identical to the sequence described by Edman et al. The actin gene isolated by Huber et al. was located by PFGE on another chromosomal band that did not contain the RP-L21 gene.

The Chornobyl accident and cognitive functioning: a follow-up study of infant evacuees at age 19 years.

BACKGROUND: The cognitive and academic outcomes of infants exposed to radiation after the meltdown at Chornobyl have been intensely debated. Western-based investigations indicate that no adverse effects occurred, but local studies reported increased cognitive impairments in exposed compared with non-exposed children. Our initial study found that at age 11 years, school grades and neuropsychological performance were similar in 300 children evacuated to Kiev as infants or in utero compared with 300 classmate controls, yet more evacuee mothers believed that their children had memory problems. This study re-examined the children's performance and academic achievement at age 19 years. METHOD: In 2005-2006, we conducted an 8-year follow-up of the evacuees (n=265) and classmate controls (n=261) assessed in Kiev in 1997. Outcomes included university attendance, tests of intelligence, attention, and memory, and subjective appraisals of memory problems. Scores were standardized using a local population-based control group (n=327). Analyses were stratified by parental education. RESULTS: Evacuees and classmates performed similarly and in the normal range on all tests, and no differential temporal changes were found. The results were comparable for the in utero subsample. The rates of university attendance and self-reported memory problems were also similar. Nevertheless, the evacuee mothers were almost three times as likely to report that their children had memory problems compared with controls. CONCLUSIONS: Chornobyl did not influence the cognitive functioning of exposed infants although more evacuee mothers still believed that their offspring had memory problems. These lingering worries reflect a wider picture of persistent health concerns as a consequence of the accident.

Cloning of DNA complementary to the measles virus mRNA encoding nucleocapsid protein.

Double-stranded cDNA synthesized from total poly(A)-containing mRNA, extracted from monkey cells infected with measles virus, has been inserted into Pst cleavage site of Escherichia coli plasmid pBR322 and cloned. A clone containing measles virus DNA sequences was identified by hybridization to a measles virus-specific 32P-labeled cDNA probe prepared from the mRNA of measles virus-infected cells. Cellular sequences in the probe were neutralized by prehybridization with an excess of unlabeled mRNA from uninfected monkey cells. The insert of cloned cDNA isolated contans 1420 base pairs, as shown by agarose gel electrophoresis and electron microscopy. The size of the mRNA complementary to this cloned cDNA is 1750 nucleotides, as determined by the reverse Southern technique. The cloned DNA fragment was further identified as the reverse transcript of the mRNA coding for the nucleocapsid protein of measles virus on the basis that the major cell-free translation product of mRNA selected by hybridization to the cloned DNA comigrated with the nucleocapsid protein and was immunoprecipitated by measles virus-specific antibodies. Subsequently, the cloned DNA was used to detect specific measles virus sequences in the poly(A)-RNA extracted from brain autopsy material from a patient with subacute sclerosing panecephalitis. The cloned DNA can thus be used as a probe to study the structure and expression of the measles genome, and in particular, to study diseases of the central nervous system in which persistent infection with measles virus has been implicated.

Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells.

The vaccinia virus/bacteriophage T7 hybrid transient expression system employs a recombinant vaccinia virus that encodes the T7 RNA polymerase gene, a plasmid vector with a gene of interest regulated by a T7 promoter, and any cell line suitable for infection and transfection. Although high expression in a majority of cells is achieved, the severe cytopathic effects of vaccinia virus and the safety precautions required for use of infectious agents are undesirable features of the system. Here, we report the construction of a highly attenuated and avian host-restricted vaccinia virus recombinant that encodes the T7 RNA polymerase gene (MVA/T7 pol) and demonstrate the use of the virus for transient expression in mammalian cells. MVA/T7 pol has reduced cytopathic effects compared to the previously used replication-competent vaccinia virus, while providing a high level of gene expression in multiple mammalian cell lines.

Coral-host specificity of Red Sea Lithophaga bivalves: interspecific and intraspecific variation in 12S mitochondrial ribosomal RNA.

Comparison of 12S mitochondrial ribosomal DNA sequences was used to approach the question of species specificity between boring bivalves of the genus Lithophaga and their coral hosts. A 450-bp long fragment was amplified by PCR from 13 individuals belonging to five subgroups of Lithophaga bivalves. These subgroups are defined according to their coral hosts species, and they belong to three currently recognized species: L. lessepsiana (1 host), L. simplex (2 hosts), and L. purpurea (2 hosts). All bivalves were collected from corals growing within an approximately 200-m section of the reef of Eilat, Red Sea. Sequence variation between members of the same species inhabiting different hosts (30-32%) was found to be very similar to the variation exhibited between recognized species. These results, when interpreted together with previously published data concerning variations among Lithophaga subgroups, support the notion of a very high degree of species specificity between Lithophaga bivalves and their coral hosts in the Red Sea.

Expression of Erythrina corallodendron lectin in Escherichia coli.

The cDNA of the Erythrina corallodendron lectin (ECorL) has been expressed in Escherichia coli. For this purpose, an NcoI site was inserted into the cDNA coding for the lectin precursor [Arango, R., Rozenblatt, S. & Sharon, N. (1990) FEBS Lett. 264, 109-112] immediately before the codon GTG (103-105) which codes for the N-terminal valine of the mature lectin. This introduced an ATG codon for a methionine preceding the valine. The mutated cDNA was ligated into pUC-8, then subcloned into the expression vector pET-3d, which carries a strong promoter derived from gene 10 of the phage T7. The recombinant plasmid was introduced into the E. coli lysogenic strain BL21(DE3). Recombinant ECorL was expressed by growing the bacteria in the presence of isopropyl beta-D-thiogalactopyranoside. Most of the recombinant lectin was found in an insoluble aggregated form as inclusion bodies and only a small part was in the culture medium in a soluble active form. Functional recombinant lectin was recovered from the inclusion bodies by solubilization with 6 M urea in cyclohexylaminopropane sulfonate pH 10.5, renaturation by 10-fold dilution in the same buffer and further adjustment of the pH to 8.0. The recombinant lectin, obtained at a yield of 4-7 mg/l culture, had, by gel filtration, a slightly lower molecular mass (56 kDa) than the native lectin, and was devoid of covalently linked carbohydrate; it was, however, essentially indistinguishable from native ECorL by other criteria, including its dimeric structure, Western blot analysis with anti-ECorL polyclonal and monoclonal antibodies, and Ouchterlony double-diffusion analysis with polyclonal antibodies, as well as hemagglutinating activity and specificity for mono- or disaccharides.

Localization of measles virus nucleic acid sequences in infected cells by in situ hybridization.

Vero cells were infected with measles virus and hybridized in situ to a cloned DNA fragment containing specific sequences for measles nucleocapsid protein. The DNA was labelled with tritium by nick-translation. The viral RNA were detected in the cytoplasm 21 hrs after infection. In many cells, the probe hybridized to nuclear structures, and in several mitotic cells, to chromosomes. After 36 hrs of infection, hybridization sites were found both in the center and in many nuclei of all the polykaryons. These results indicate that cellular distribution of viral RNA molecules varies in the course of infection. They further suggest that the nucleus plays a more active role than expected in measles virus transcription and replication.

Infective substructures of measles virus from acutely and persistently infected cells.

Ribonucleoprotein from cells acutely or persistently infected with measles virus were shown to be infectious by the calcium phosphate technique. Very little or no infectivity was obtained when calcium phosphate precipitation was omitted. Electron microscopy showed that the majority of ribonucleoprotein structures isolated from acutely infected cells were folded, whereas those from persistently infected cells were linear in appearance.

Characterization of measles virus-specific proteins synthesized in vivo and in vitro from acutely and persistently infected cells.

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.

Synthesis of soybean agglutinin in bacterial and mammalian cells.

The cDNA of soybean agglutinin (SBA), a glycoprotein lectin, obtained from the mRNA of soybean seeds at mid-maturation, was cloned in a lambda gt 10 phage and subcloned in a pUC-8 plasmid. Probing with a fragment of the lectin gene [Vodkin, L. O., Rhodes, P. R. & Goldberg, R. B. (1983) Cell 34, 1023-1031] afforded a clone of 1012 nucleotides containing the complete coding region of 858 nucleotides for the precursor to soybean agglutinin. The deduced amino acid sequence contains the 253 residues of the mature lectin and an hydrophobic N-terminal signal peptide of 32 amino acids. Expression in Escherichia coli of the cDNA coding for the precursor to the lectin or for the mature lectin led to the accumulation of large quantities of inclusion bodies, from which mature SBA was isolated in small yield (up to 1 mg/l). It was identical with the native lectin in the hemagglutinating activity and carbohydrate specificity, N-terminal sequence and oligomeric structure, but, because it was not glycosylated, its subunit mass was lower by 2 kDa. Our findings show that pre-SBA is processed into the mature form in the bacteria, and that, contrary to what has been suggested [Nagai, K. & Yamaguchi, H. (1993) J. Biochem. (Tokyo) 113, 123-125], glycosylation is not essential for the folding of the lectin, nor for its subunit assembly into a biologically active tetramer. To obtain recombinant SBA in secreted form, the pre-SBA cDNA was subcloned in pTM1 vector and the construct inserted into vaccinia virus. When monkey BS-C-1 cells were infected by the virus, using a double expression protocol, recombinant lectin was secreted into the growth medium, from which it was isolated by immunoaffinity chromatography at a yield similar to that from the bacteria. Except for its lower hemagglutinating activity, the product was indistinguishable from native SBA in all properties tested. It was also susceptible to digestion by endo-beta-N-acetylglucosaminidase H or N-glycanase which caused a decrease of 2 kDa in its subunit mass and gave the same results on lectin blot analysis, indicating that it too is a glycoprotein with a single oligomannose unit.

Cloning and characterization of DNA complementary to the measles virus mRNA encoding hemagglutinin and matrix protein.

Since cloning and characterization of DNA complementary to measles virus mRNA encoding for the nucleocapsid protein (M. Gorecki and S. Rozenblatt, Proc, Natl. Acad, Sci, U.S.A. 77:3686--3690, 1980), two additional measles-specific clones containing different classes of sequences have been characterized. The cloned plasmids contain inserts of 480 and 530 base pairs as shown by agarose gel electrophoresis and electron microscopy. The sizes of the mRNA species complementary to these inserts are 1,700 and 1,550 nucleotides, respectively as determined by the Northern technique. The cloned DNA fragments were further identified as reverse transcripts of the mRNA coding for the glycoprotein and matrix protein of measles virus. The major cell-free translation products of mRNA selected by hybridization to the individual cloned DNAs comigrated with the 70K in vitro products and matrix proteins. One of the cell-free translation products (70K) was also immunoprecipitated specifically with monoclonal antibodies against measles virus glycoprotein.