Protective immune responses induced by vaccination with an expression genomic library of Leishmania major.
(41/2024)
To develop an effective vaccine against the intracellular protozoan parasite Leishmania spp., we investigated the feasibility of expression library immunization (ELI) in the mouse. Genomic expression libraries of L. major were constructed and used to immunize mice. One of the three libraries (L1, with 10(5) clones) induced a significant protective immune response and delayed the onset of lesion development in highly susceptible BALB/c mice after i.m. immunization, compared with control mice immunized with the empty vector (EV). L1 was then divided into five sublibraries of approximately 2 x 10(4) clones each. Mice immunized with one of the sublibraries (SL1A) developed an even stronger protective effect than that induced by L1. SL1A was further divided into 20 sublibraries (SL2) of approximately 10(3) clones each. One of the SL2 libraries (SL2G) induced a strong protective effect against L. major infection. In direct comparative studies, the protective effect of the sublibraries was in the order of SL2G > SL1A > L1. Lymphoid cells from mice vaccinated with SL2G produced more IFN-gamma and NO, compared with cells from control mice injected with EV. Serum from the vaccinated mice also contained more parasite-specific IgG2a Ab, compared with controls. Therefore, these data demonstrate that ELI is feasible against this complex intracellular parasitic infection, by preferentially inducing the development of Th1 responses. Furthermore, by sequential division of the libraries, this approach may be used to enrich and identify protective genes for effective gene vaccination against other parasitic infections. (+info)
Characterization of an atypical superoxide dismutase from Sinorhizobium meliloti.
(42/2024)
Sinorhizobium meliloti Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a sod fragment amplified by PCR from degenerate primers as a probe. The sodA gene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical M(r) of 22,430 and pI of 5. 8. S. meliloti SOD complemented a deficient E. coli mutant, restoring aerobic growth of a sodA sodB recA strain, when the gene was expressed from the synthetic tac promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H(2)O(2). The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer). The apoenzyme was prepared from crude extracts of S. meliloti. Activity was restored by dialysis against either MnCl(2) or Fe(NH(4))(2)(SO(4))(2), demonstrating the cambialistic nature of the S. meliloti SOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H(2)O(2). Sequence comparison with 70 FeSODs and MnSODs indicates that S. meliloti SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H(2)O(2) of this unusual Fe-type SOD. (+info)
Molecular cloning, sequence analysis, and characterization of a penicillin-resistant DD-carboxypeptidase of Myxococcus xanthus.
(43/2024)
We have cloned a gene, pdcA, from the genomic library of Myxococcus xanthus with an oligonucleotide probe representing conserved regions of penicillin-resistant DD-carboxypeptidases. The amino- and carboxy-terminal halves of the predicted pdcA gene product showed significant sequence similarity to N-acetylmuramoyl-L-alanine amidase and penicillin-resistant DD-carboxypeptidase, respectively. The pdcA gene was expressed in Escherichia coli, and the characteristics of the gene product were similar to those of DD-carboxypeptidase (VanY) of vancomycin-resistant enterococci. No apparent changes in cell growth, sporulation, or germination were observed in pdcA deletion mutants. (+info)
Identification and sequence analysis of the Marek's disease virus serotype 2 gene homologous to the herpes simplex virus type 1 UL52 protein.
(44/2024)
The gene of Marek's disease virus serotype 2 (MDV2) homologous to the UL52 gene of herpes simplex virus type 1 (HSV-1) was identified and characterized. The MDV2 UL52 homologous gene encodes 1,071 amino acids with a molecular weight of 118.7 kDa, which includes putative metal-binding site and overlapping region with the UL53 homologous gene. Although a putative polyadenylation signal sequence was found in the downstream of the MDV2 UL52 gene, a MDV2 UL52 DNA probe reacted only with the polycistronic 6.3 kb transcript, representing the UL52 and the downstream genes of UL53 and UL54. Transcriptional pattern of this region of MDV2 was somewhat different from corresponding regions of HSV-1 and infectious laryngotracheitis virus. (+info)
Complete sequence of the 23-kilobase human COL9A3 gene. Detection of Gly-X-Y triplet deletions that represent neutral variants.
(45/2024)
We report the complete sequence of the human COL9A3 gene that encodes the alpha3 chain of heterotrimeric type IX collagen, a member of the fibril-associated collagens with interrupted triple helices family of collagenous proteins. Nucleotide sequencing defined over 23,000 base pairs (bp) of the gene and about 3000 bp of the 5'-flanking sequences. The gene contains 32 exons. The domain and exon organization of the gene is almost identical to a related gene, the human COL9A2 gene. However, exon 2 of the COL9A3 gene codes for one -Gly-X-Y- triplet less than exon 2 of the COL9A2 gene. The difference is compensated by an insertion of 9 bp coding for an additional triplet in exon 4 of the COL9A3 gene. As a result, the number of -Gly-X-Y- repeats in the third collagenous domain remains the same in both genes and ensures the formation of an in-register triple helix. In the course of screening this gene for mutations, heterozygosity for separate 9-bp deletions within the COL1 domain were identified in two kindreds. In both instances, the deletions did not co-segregate with any disease phenotype, suggesting that they were neutral variants. In contrast, similar deletions in triple helical domain of type I collagen are lethal. To study whether alpha3(IX) chains with the deletion will participate in the formation of correctly folded heterotrimeric type IX collagen, we expressed mutant alpha3 chains together with normal alpha1 and alpha2 chains in insect cells. We show here that despite the deletion, mutant alpha3 chains were secreted as heterotrimeric, triple helical molecules consisting of three alpha chains in a 1:1:1 ratio. The results suggest that the next noncollagenous domain (NC2) is capable of correcting the alignment of the alpha chains, and this ensures the formation of an in-register triple helix. (+info)
Functional genomics.
(46/2024)
Complete genome sequences are providing a framework to allow the investigation of biological processes by the use of comprehensive approaches. Genome analysis also is having a dramatic impact on medicine through its identification of genes and mutations involved in disease and the elucidation of entire microbial gene sets. Studies of the sequences of model organisms, such as that of the nematode worm Caenorhabditis elegans, are providing extraordinary insights into development and differentiation that aid the study of these processes in humans. The field of functional genomics seeks to devise and apply technologies that take advantage of the growing body of sequence information to analyze the full complement of genes and proteins encoded by an organism. (+info)
The poly(A)-limiting element is a conserved cis-acting sequence that regulates poly(A) tail length on nuclear pre-mRNAs.
(47/2024)
Most vertebrate mRNAs exit the nucleus with a 200+-residue poly(A) tail and are deadenylated to yield heterogeneous polymers of 50-200 adenosine residues on any given mRNA. We previously reported that Xenopus albumin mRNA and pre-mRNA have an unusually short, discrete 17-residue poly(A) tail and showed that regulation of poly(A) length is controlled independently by two cis-acting poly(A)-limiting elements (PLE A and PLE B) located in the terminal exon. The present study sought to determine the generality of this regulatory mechanism. Transferrin mRNA also has a discrete <20-nt poly(A) tail, and deletion mapping experiments identified an element homologous to the albumin gene PLE B within the terminal exon of the transferrin gene that conferred poly(A) length regulation on a globin reporter mRNA. Based on this similarity the PLE B sequence was used in a database search to identify candidate mRNA targets for regulated polyadenylation. Of the several hundred sequences identified in this manner we focused on HIV-EP2/Schnurri-2, a member of a family of genes encoding related zinc finger transcription factors. A striking feature of the PLE-like element in these genes is its location 10-33 bp upstream of the translation stop codon. We demonstrate that HIV-EP2 mRNA has a <20-nt poly(A) tail, for which the identified PLE-like sequence is responsible. These results indicate that the presence of a PLE can predict mRNAs with <20-nt poly(A) tails, and that nuclear regulation of poly(A) tail length is a feature of many mRNAs. (+info)
A vital role for glycosphingolipid synthesis during development and differentiation.
(48/2024)
Glycosphingolipids (GSLs) are believed to be integral for the dynamics of many cell membrane events, including cellular interactions, signaling, and trafficking. We have investigated their roles in development and differentiation by eliminating the major synthesis pathway of GSLs through targeted disruption of the Ugcg gene encoding glucosylceramide synthase. In the absence of GSL synthesis, embryogenesis proceeded well into gastrulation with differentiation into primitive germ layers and patterning of the embryo but was abruptly halted by a major apoptotic process. In vivo, embryonic stem cells deficient in GSL synthesis were again able to differentiate into endodermal, mesodermal, and ectodermal derivatives but were strikingly deficient in their ability to form well differentiated tissues. In vitro, however, hematopoietic and neuronal differentiation could be induced. The results demonstrate that the synthesis of GSL structures is essential for embryonic development and for the differentiation of some tissues and support the concept that GSLs are involved in crucial cell interactions mediating these processes. (+info)