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PubMed1997

Proteome research: complementarity and limitations with respect to the RNA and DNA worlds.

A methodological overview of proteome analysis is provided along with details of efforts to achieve high-throughput screening (HTS) of protein samples derived from two-dimensional electrophoresis gels. For both previously sequenced organisms and those lacking significant DNA sequence information, mass spectrometry has a key role to play in achieving HTS. Prototype robotics designed to conduct appropriate chemistries and deliver 700-1000 protein (genes) per day to batteries of mass spectrometers or liquid chromatography (LC)-based analyses are well advanced, as are efforts to produce high density gridded arrays containing > 1000 proteins on a single matrix assisted laser desorption ionisation/time-of-flight (MALDI-TOF) sample stage. High sensitivity HTS of proteins is proposed by employing principally mass spectrometry in an hierarchical manner: (i) MALDI-TOF-mass spectrometry (MS) on at least 1000 proteins per day; (ii) electrospray ionisation (ESI)/MS/MS for analysis of peptides with respect to predicted fragmentation patterns or by sequence tagging; and (iii) ESI/MS/MS for peptide sequencing. Genomic sequences when complemented with information derived from hybridisation assays and proteome analysis may herald in a new era of holistic cellular biology. The current preoccupation with the absolute quantity of gene-product (RNA and/or protein) should move backstage with respect to more molecularly relevant parameters, such as: molecular half-life; synthesis rate; functional competence (presence or absence of mutations); reaction kinetics; the influence of individual gene-products on biochemical flux; the influence of the environment, cell-cycle, stress and disease on gene-products; and the collective roles of multigenic and epigenetic phenomena governing cellular processes. Proteome analysis is demonstrated as being capable of proceeding independently of DNA sequence information and aiding in genomic annotation. Its ability to confirm the existence of gene-products predicted from DNA sequence is a major contribution to genomic science. The workings of software engines necessary to achieve large-scale proteome analysis are outlined, along with trends towards miniaturisation, analyte concentration and protein detection independent of staining technologies. A challenge for proteome analysis into the future will be to reduce its dependence on two-dimensional (2-D) gel electrophoresis as the preferred method of separating complex mixtures of cellular proteins. Nonetheless, proteome analysis already represents a means of efficiently complementing differential display, high density expression arrays, expressed sequence tags, direct or subtractive hybridisation, chromosomal linkage studies and nucleic acid sequencing as a problem solving tool in molecular biology.

Animals↗

PubMed1994

A new genetically engineered vaccine for animal growth promotion.

The chemically synthesized somatostatin (ss) gene was fused in phase with the 3'-end of hepatitis B virus surface antigen (HBsAg) gene. The fusion gene HBs/ss was then recombined into the genome of vaccinia virus. This recombinant virus (vv-HBs/ss) can express hybrid HBsAg/ss particles which present as determinants on their surfaces, thereby bearing a good immunogenicity. This new strategical vaccine of ss can elicit the production of antibody capable of neutralizing ss in the plasma, and consequently enhance the growth of animals.

Animals↗

PubMed2004

Transformation of Saussurea medusa for hairy roots and jaceosidin production.

Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A. rhizogenes strain R1601 in N6 medium. PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A. rhizogenes into the genome of S. medusa hairy roots. In N6 medium, maximum biomass of the hairy root cultures was achieved [8 g (dry weight) per liter; growth ratio 35-fold] after 21 days of culture. The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture. The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium. In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.

Agriculture↗

PubMed2002

[Delivery of secreted placental alkaline phosphatase (SEAP) gene in vitro and in vivo as a component of recombinant avian adenovirus (CELO)].

Recombinant adenoviruses capable of expressing the gene of secreted placentary alkaline phosphatase (SEAP) under control of CMV-promoter was obtained on the basis of CELO avian adenovirus and human adenovirus-5 (Ad5) genomes. The efficiency of the CELO vector was determined in experiments with transduction of human (293, A549, and H1299), mouse (B16), and avian (LMH) cell cultures. It was shown in C57BL/6 mice in vivo that SEAP gene is expressed under conditions of intravenous, intranasal, and intratumoral application of recombinant adenovirus CELO-SEAP. The duration of expression of the alkaline phosphatase CELO = SEAP gene in immunocompetent mouse body was 21 days. The level of SEAP gene expression was measured in the allantois fluid of chicken embryo infected with recombinant adenovirus CELO-SEAP.

Alkaline Phosphatase↗

PubMed1990

Factors affecting efficient infection of tobacco with in vitro RNA transcripts from cloned cDNAs of satellite tobacco mosaic virus.

Recombinant cDNA clones of the complete satellite tobacco mosaic virus (STMV) genome (1059 ribonucleotides) were constructed with unique Xbal and HindIII or Pstl restriction sites engineered at the 5' and 3' termini, respectively. The genome-length cDNAs were positioned downstream of T7 or SP6 phage promoters. Genome-sense RNAs transcribed in vitro from the T7 promoter were biologically active, while negative-sense RNAs transcribed in vitro from the SP6 promoter were not. Constructs that were identical to STMV and two other constructs in which there were two or six specific nucleotide differences in the 3' noncoding region yielded RNAs that were infectious. Sequence analysis of the progeny RNA derived from infections with transcripts containing nucleotide differences between nucleotides 682 and 753 revealed that these changes in sequence were maintained. In contrast, differences in the nucleotide sequence between nucleotides 989 and 1059 were not maintained in progeny RNA; one mutant reverted to the wild-type sequence, and the other generated a new sequence during infection.

Base Sequence↗

PubMed2003

Plant molecular biology and biotechnology research in the post-recombinant DNA era.

After the beginning of the recombinant DNA era in the mid-1970s, researchers in India started to make use of the new technology to understand the structure of plant genes and regulation of their expression. The outcome started to appear in print in early the 1980s and genes for histones, tubulin, photosynthetic membrane proteins, phototransduction components, organelles and those regulated differentially by developmental and extrinsic signals were sequenced and characterized. Some genes of biotechnological importance like those encoding an interesting seed protein and the enzyme glyoxalase were also isolated. While work on the characterization of genome structure and organization was started quite early, it remained largely focused on the identification of DNA markers and genetic variability. In this context, the work on mustard, rice and wheat is worth mentioning. In the year 2000, India became a member of the international consortium to sequence entire rice genome. Several laboratories have also given attention to regulated expression of plastid and nuclear genes as well as to isolate target-specific promoters or design promoters with improved potential. Simultaneously, transgenic systems for crops like mustard, rice, wheat, cotton, legumes and several vegetables have been established. More recently, genes of agronomic importance like those for insect resistance, abiotic stress tolerance, nutritional improvement and male sterility, isolated in India or abroad, have been utilized for raising transgenics for crop improvement. Some of these transgenics have already shown their potential in containment facility or limited field trials conducted under the stipulated guidelines. Plant molecular biology and biotechnology are thus clearly poised to make an impact on research in basic biology and agriculture in the near future.

Agriculture↗

PubMedDate not supplied

[Molecular biology at the service of the daily medical virology. 1. Methodological principles].

Molecular biology techniques which can rapidly detect a few copy number of viral genome allow to obtain new parameters for the diagnosis, the prognosis, and the monitoring of viral diseases in addition to or in substitution for cell culture and antigen and antibody detection. The methods are based on the virus structural organization and on their replication into cells. They use the tools which have been developed to analyse nucleic acids. Molecular hybridization (dot-blot, southern-blot, liquid phase hybridization, in situ hybridization), enzymatic amplification (PCR, nested-PCR, RT-PCR, NASBA, bDNA, multiplex PCR), DNA and RNA quantification (quantitative PCR or RT-PCR, NASBA, bDNA) and genotyping (virus types, mutations, variant virus) are used. Nucleic acid extracts native or amplified with a reporter molecule (dUTP-biotin or dUTP-digoxigenin) are identified using gel electrophoresis followed by hybridization or using microwell capture hybridization assay and colorimetric immunoenzymatic or bioluminescence assays.

Genetic Engineering↗

PubMed2026

Unlocking the molecular engineering of Geobacillus glycoside hydrolases as a source of industrial biocatalysts.

This review examines Geobacillus sensu stricto as a source of thermostable glycoside hydrolases (GH) for biomass conversion, food processing, and enzyme engineering. Recent peer-reviewed literature was assessed with emphasis on taxonomy, genome-based Carbohydrate-Active Enzymes (CAZyme) prediction, biochemical validation, structural data, and engineering case studies. Taxonomic boundaries were interpreted using current Anoxybacillaceae frameworks, with Parageobacillus treated as a related comparator rather than as Geobacillus. The strongest evidence supports GH13 alpha-amylases, xylan-active systems, beta-xylosidases, and selected accessory enzymes. Recent studies also show that genome mining must be coupled with enzymatic assays and product profiling because CAZyme annotation alone does not prove industrial function. Molecular engineering has improved relevant traits, including the longer thermal half-life of engineered G. stearothermophilus alpha-amylase variants, the increased catalytic efficiency of oligo-alpha-1,6-glucosidase variants, and improved AmyS expression in Bacillus subtilis. Geobacillus glycoside hydrolases are best interpreted as process-specific, engineerable biocatalytic templates. Their translation requires reliable taxonomy, functional validation, structural interpretation, scalable expression and testing on realistic substrates. This synthesis also recognises current limitations: many predicted CAZymes still lack biochemical validation, complete cellulolytic systems remain less mature than xylan- and starch-active systems, and scale-up data remain scarce.

Geobacillus↗

PubMed2002

Human l1 retrotransposition is associated with genetic instability in vivo.

Retrotransposons have shaped eukaryotic genomes for millions of years. To analyze the consequences of human L1 retrotransposition, we developed a genetic system to recover many new L1 insertions in somatic cells. Forty-two de novo integrants were recovered that faithfully mimic many aspects of L1s that accumulated since the primate radiation. Their structures experimentally demonstrate an association between L1 retrotransposition and various forms of genetic instability. Numerous L1 element inversions, extra nucleotide insertions, exon deletions, a chromosomal inversion, and flanking sequence comobilization (called 5' transduction) were identified. In a striking number of integrants, short identical sequences were shared between the donor and the target site's 3' end, suggesting a mechanistic model that helps explain the structure of L1 insertions.

5' Flanking Region↗

PubMedDate not supplied

A restriction cleavage and transfection system for introducing foreign DNA sequences into the genome of a herpesvirus.

This report describes a simple and efficient system for construction of recombinant pseudorabies (Aujeszky's disease) virus (PrV) which is based on the use of a unique restriction site inserted into the viral genome. This system enables the recovery of genetically modified viruses without screening or selection for a specific phenotype, since practically all mature viral particles obtained carry the foreign sequences. To demonstrate, we introduced the tumour suppressor protein-53 (p53) gene into two different intergenic locations of PrV: the ribonucleotide reductase (rr) gene and the promoter of a putative latency gene (PLAT), located at the inverted repeat (IR) region of the viral genome. As a first step, we engineered a unique EcoRI recognition site into the rr gene or into both copies of PLAT with the help of marker transfer using the bacterial lacZ gene. Then, in both cases viral DNAs were cut with the restriction endonuclease EcoRI followed by treatment with calf intestinal phosphatase and used for cotransfection into porcine kidney cells with a plasmid containing the p53 gene flanked by viral DNAs homologous to the target region. As a result of this process, in most of the experiments, we obtained recombinant viruses without the background of parental viruses. Here we show that this method can be used for directional insertion of exogenous sequences into either the unique or the IR region of the PrV chromosome. In principle, this system should be applicable to the construction of recombinant derivatives of any viruses having infectious DNA.

Animals↗

PubMed1999

Zinc finger peptides for the regulation of gene expression.

Zinc fingers are small DNA-binding peptide motifs that were discovered in this laboratory. These motifs can be used as modular building blocks for the construction of larger protein domains that recognise and bind to specific DNA sequences. Phage display has been used to create a large library of different zinc fingers from which selections were made for binding to a given DNA sequence. From this database there have been elucidated elements of recognition rules that relate the amino acid sequence of a finger to its preferred DNA binding site. Control of gene expression using designed zinc finger peptides has been demonstrated by the specific inhibition of an oncogene mouse cell line and also by switching on genes in expression plasmids. These experiments demonstrate that zinc finger DNA-binding domains can be engineered de novo to recognise given DNA sequences. Five to six individual zinc fingers linked together would recognise a DNA sequence 15-18 bp in length, sufficiently long to constitute a rare address in the human genome. By adding functional groups to the engineered DNA-binding domains, e.g. silencing domains, novel transcription factors can be generated to up- or downregulate expression of a target gene. Among potential applications are the repression of oncogene expression and the disruption of the reproductive cycle of virus infection.

Animals↗

PubMed2000

Mammalian genomes contain active recombinase recognition sites.

Recombinases derived from microorganisms mediate efficient site-specific recombination. For example, the Cre recombinase from bacteriophage P1 efficiently carries out recombination at its loxP target sites. While this enzyme can function in mammalian cells, the 34bp loxP site is expected to be absent from mammalian genomes. We have discovered that sequences from the human and mouse genomes surprisingly divergent from loxP can support Cre-mediated recombination at up to 100% of the efficiency of the native loxP site in bacterial assays. Transient assays in human cells demonstrate that such pseudo-lox sites also support Cre-mediated integration and excision in the human cell environment. Pseudo sites for Cre and other recombinases may be useful for site-specific insertion of exogenous genes into mammalian genomes during gene therapy and other genetic engineering processes.

Animals↗

PubMed1993

Assembly of functional Sindbis virus RNA replication complexes: requirement for coexpression of P123 and P34.

A vaccinia virus transient expression system was used to determine which of the Sindbis virus (SIN) proteins and/or polyproteins are necessary for the formation of active replication complexes and, in particular, to analyze the role of nsP4, the putative polymerase, versus P34 in RNA replication. We generated vaccinia virus recombinants in which the cDNA for the entire SIN nonstructural coding region as well as cDNA copies of the individual nonstructural proteins (nsPs) and several intermediate polyproteins were placed downstream of the promoter for T7 RNA polymerase and the encephalomyocarditis virus 5' untranslated region. The proteins expressed by the vaccinia virus recombinants comigrate with authentic proteins synthesized in SIN-infected cells, and the polyproteins appear to be processed to the individual proteins of the correct size. To examine the replication efficiencies of different protein combinations, a vaccinia virus recombinant was designed to express an engineered substrate RNA which could serve as a template for replication and subgenomic mRNA transcription by the SIN nsPs. Expression of the entire SIN nonstructural coding region resulted in the synthesis of high levels of both genomic and subgenomic RNAs derived from the engineered template. No RNA replication could be detected during coexpression of the four individual nsPs, although the proteins were indistinguishable, in terms of electrophoretic mobility, from those synthesized in SIN-infected cells. Coexpression of polyproteins P12, P23, and/or P34 with the individual nsPs also did not result in detectable levels of RNA replication. However, when P123 and P34 were coexpressed, efficient RNA replication and subgenomic mRNA transcription of the substrate RNA was observed. Coexpression of nsP4 with P123 resulted in the synthesis of only minus-strand RNAs. These studies show that expression of both P123 and P34 is necessary for establishment of functional RNA replication and transcription complexes and raise the possibility that the polyproteins themselves may be functional components of these complexes. In addition, these data indicate that an nsP4 moiety expressed independently with an additional N-terminal methionine is capable of functioning in minus-strand but not plus-strand RNA synthesis.

Animals↗

PubMed2006

Circumventing recombination events encountered with production of a clinical-grade adenoviral vector with a double-expression cassette.

Delivery of multiple exogenous genes into target cells is important for a broad range of gene therapy applications, including combined therapeutic gene expression and noninvasive imaging. Previous studies ( Mol Ther 4: 223-231, 2001 ) have described the adenoviral vector RGDTKSSTR with a double-expression cassette that encodes herpes simplex virus thymidine kinase (HSVtk) for molecular chemotherapy and human somatostatin receptor subtype-2 (hSSTR2) for indirect imaging. In this vector, both genes are inserted in place of the E1 region of the adenoviral genome and expressed independently from two cytomegalovirus (CMV) promoters. During production of clinical-grade RGDTKSSTR, we found that the CMV promoters and simian virus 40 (SV40) poly(A) regions located in both expression cassettes provoked homologous recombination and deletion of one of the cassettes. To resolve this problem, we designed a strategy for substituting the duplicate promoters and poly(A) regions. We placed the hSSTR2 gene in the new Ad5.SSTR/TK.RGD vector under the control of a CMV promoter with a bovine growth hormone poly(A) region, whereas the SV40 promoter, enhancer, and poly(A) signal controlled HSVtk expression. This use of different regulatory sequences allowed independent expression of both transgenes from a single adenoviral vector and circumvented the recombination problem. Reconstruction of the vector with a double-expression cassette enables its use in human clinical trials.

Adenoviridae↗

PubMed1995

Infectious transcripts of tick-borne encephalitis virus, generated in days by RT-PCR.

Construction of infectious clones of flaviviruses can be problematic owing to instability, toxicity, and recombination events occurring while cloning cDNA in the bacterial vectors. To overcome these difficulties we have devised a rapid and simple method for producing an infectious genetically engineered tick-borne encephalitis virus in less than 10 days using viral RNA from an unpurified virus suspension. The experimental protocol utilized the high fidelity reverse transcription-polymerase chain reaction to produce two long (5.7 and 5.2 kb) overlapping cDNA segments. To produce full-length cDNA the two overlapping segments were either ligated or fused by polymerase chain reaction. The cDNA was then transcribed and the derived full-length RNA was injected intracerebrally into young mice which reproduced the infectious virus within 8-20 days. To differentiate the engineered virus from parent virus, a Sunl restriction site was introduced by substituting nucleotides at positions 5688 and 5691 of the viral genome. This restriction site was present in the engineered virus recovered from infected mice. Antigenic and electrophoretic analysis of the proteins recovered from the engineered virus confirmed that it was indistinguishable from parent virus. In addition to its applicability as a rapid method of producing infectious engineered virus, this protocol offers the opportunity to introduce changes by site-directed mutagenesis without needing to clone the viral DNA. The method should be applicable to most viruses possessing an infectious RNA molecule and reduces the time required to produce a genetically engineered virus from years to days. When appropriate, the choice of mice for transfection of RNA has the advantage of being extremely simple, very sensitive, and producing high titers of stable virus.

Animals↗

PubMed1994

A genetic selection method for the transfer of HSV-1 glycoprotein B mutations from plasmid to the viral genome: preliminary characterization of transdominance and entry kinetics of mutant viruses.

A genetic selection procedure has been devised to transfer mutations from a plasmid to the viral genome. Reagents were constructed so that the recombination events that occur during cotransfection rescue a mutation in a recipient viral genome and, simultaneously, transfer a mutation in an adjacent (target) gene to the viral genome. In the example presented here the two adjacent genes are essential for viral replication, so that a transformed cell line that expresses both genes and another cell line that expresses only the target gene are required. Ideally the recipient viral genome should be deleted for the entire target gene, and the deletion should extend a short distance into the adjacent gene. In the present study UL27 (glycoprotein gB) of herpes simplex virus type 1 (KOS) is the target gene for mutation transfer and the upstream gene UL28, which specifies the ICP18.5 polypeptide, is the marker-rescue gene. A recipient virus that was deleted for DNA sequences encoding the C-terminal 74 residues of ICP18.5 and the N-terminal 711 residues of gB was constructed. All of the gB mutant plasmids used overlap the ICP18.5 deletion, and therefore, recombination events that rescue the ICP18.5 deletion must transfer a gB mutation present in codons 1-711 of the rescuing plasmid. Recombinant viruses that contain the gB mutation, unlike the parental gB-/18.5- virus, will grow on cells that express only gB (D6). This procedure has been used to transfer insertion, deletion, and chain termination mutations into the gB gene of the KOS genome. Southern blot analysis confirmed the transfer of the mutations to the viral genome. Analysis of radioactively labeled, infected cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), before and after immune precipitation with gB-specific antibodies, confirmed the presence of the mutant polypeptides in the lysates. The incorporation of the mutant polypeptides into the envelopes of purified released viruses was measured. The yield of plaque forming units per cell was determined from single step growth curves after infection of permissive D6 cells. Virus yield, a measure of transdominance, varied for the different mutants. The maximum reduction in virus yield observed was sixfold relative to the wild-type virus. Transdominance was a result of both the lack of glycoprotein processing and the presence of gB-oligomerization sites. A reduced yield was also observed for a previously isolated gB-null mutant virus, K082. The amount of gB produced in D6 cells, infected with K082, was less than that produced in KOS infected cells, which may account for the reduced yield.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Line↗

PubMed2003

Development of adenovirus serotype 35 as a gene transfer vector.

While 51 human adenoviral serotypes have been identified to date, the vast majority of adenoviral vectors designed for gene transfer have been generated in the adenovirus serotype 5 (Ad5) backbone. Viral infections caused by Ad5 are endemic in most human populations and the majority of humans carry preexisting humoral and/or cellular immunity to Ad5 which may severely limit the use of Ad5-based vectors for gene therapy applications. To circumvent this preexisting Ad5 immunity, we have identified Ad35 as an alternative adenoviral serotype to which the majority of humans do not have neutralizing antibodies. Importantly, Ad35 can be grown to high titers with a low particle-to-PFU ratio. As a prerequisite for the development of Ad35 for use as a gene transfer vector, a genome organization map was constructed using the available Ad35 sequence information, and E1a-deficient Ad35 vectors encoding marker genes were generated. Ad35 biodistribution in mice was assessed following intravenous administration and compared with that of Ad5. Extremely low levels of Ad35 were detected in all organs evaluated, including liver, lung, spleen, and bone marrow, while Ad5 displayed high transduction of these organs. Due to the lack of Ad35 liver tropism, minimal hepatotoxicity was observed in mice treated with Ad35. Furthermore, the half-life of Ad35 in mouse blood was found to be two to three times longer than that of Ad5. These data suggest that either mice do not express the Ad35 cell surface receptor or that Ad35 does not efficiently transduce mouse cells in vivo following systemic delivery. Therefore, to begin to elucidate the Ad35 cell entry mechanisms, in vitro competition studies were performed. These data demonstrated that Ad35 cell entry is CAR independent, and may involve protein(s) expressed on most human cells.

Adenoviridae↗

PubMed2004

Stem cell responses in tooth regeneration.

Scientific advances in the creation of restorative biomaterials, in vitro cell culture technology, tissue grafting, tissue engineering, molecular biology, and the human genome project provide the basis for the introduction of new technologies into dentistry. This review is intended to facilitate the development of stem cell therapy for use with established therapeutic modalities to restore and regenerate oral tissues. Teeth have been shown to mineralize in response to injury for many decades, but only in recent years has the position of the stem cells been localized around blood vessels. The cells have been identified as myofibroblastoid pericytes. The ability to control the differentiation and proliferation of these cells is being examined to create stem cell therapies that can solve dental problems more effectively than current treatment regimes. Although the problems of introducing these technologies are substantial, the potential benefits to patients and the profession are equally promising - a cure for caries and diseases, a cure for oral cancer, correction of congenital defects, and the regeneration of teeth and tissues to restore oral functions. The purpose of this review is to describe how these new technologies can most usefully be employed in dentistry to enable clinicians to satisfy patient demand for a nondefective dentition.

Animals↗