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At least 1,081 records · Page 60Linked to original sources
PubMed2002

A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants, Caenorhabditis elegans, and Drosophila. The discovery that synthetic double-stranded, 21-nt small interfering RNA triggers gene-specific silencing in mammalian cells has further expanded the utility of RNAi into mammalian systems. Here we report a technology that allows synthesis of small interfering RNAs from DNA templates in vivo to efficiently inhibit endogenous gene expression. Significantly, we were able to use this approach to demonstrate, in multiple cell lines, robust inhibition of several endogenous genes of diverse functions. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells.

Animals↗
PubMed2003

Protection and compensation in the influenza virus-specific CD8+ T cell response.

Influenza virus-specific CD8+ T cells generally recognize peptides derived from conserved, internal proteins that are not subject to antibody-mediated selection pressure. Prior exposure to any one influenza A virus (H1N1) can prime for a secondary CD8+ T cell response to a serologically different influenza A virus (H3N2). The protection afforded by this recall of established CD8+ T cell memory, although limited, is not negligible. Key characteristics of primary and secondary influenza-specific host responses are probed here with recombinant viruses expressing modified nucleoprotein (NP) and acid polymerase (PA) genes. Point mutations were introduced into the epitopes derived from the NP and PA such that they no longer bound the presenting H2Db MHC class I glycoprotein, and reassortant H1N1 and H3N2 viruses were made by reverse genetics. Conventional (C57BL/6J, H2b, and Ig+/+) and Ig-/- (muMT) mice were more susceptible to challenge with the single NP [HKx31 influenza A virus (HK)-NP] and PA (HK-PA) mutants, but unlike the Ig-/- mice, Ig+/+ mice were surprisingly resistant to the HK-NP/-PA double mutant. This virus was found to promote an enhanced IgG response resulting, perhaps, from the delayed elimination of antigen-presenting cells. Antigen persistence also could explain the increase in size of the minor KbPB1703 CD8+ T cell population in mice infected with the mutant viruses. The extent of such compensation was always partial, giving the impression that any virus-specific CD8+ T cell response operates within constrained limits. It seems that the relationship between protective humoral and cellular immunity is neither simple nor readily predicted.

Agammaglobulinemia↗
PubMed2001

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry.

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which "mimics" embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

Antigens, CD↗
PubMed2001

Efficient chromosomal transposition of a Tc1/mariner- like transposon Sleeping Beauty in mice.

The presence of mouse embryonic stem (ES) cells makes the mouse a powerful model organism for reverse genetics, gene function study through mutagenesis of specific genes. In contrast, forward genetics, identification of mutated genes responsible for specific phenotypes, has an advantage to uncover novel pathways and unknown genes because no a priori assumptions are made about the mutated genes. However, it has been hampered in mice because of the lack of a system in which a large-scale mutagenesis and subsequent isolation of mutated genes can be performed efficiently. Here, we demonstrate the efficient chromosomal transposition of a Tc1/mariner-like transposon, Sleeping Beauty, in mice. This system allows germ-line mutagenesis in vivo and will facilitate certain aspects of phenotype-driven genetic screening in mice.

Animals↗
PubMed2001

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.

Short interfering RNAs (siRNAs) are double-stranded RNAs of approximately 21-25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates. siRNAs have a characteristic structure, with 5'-phosphate/3'-hydroxyl ends and a 2-base 3' overhang on each strand of the duplex. In this study, we present data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice. In each case, the interference by siRNAs was superior to the inhibition of gene expression mediated by single-stranded antisense oligonucleotides. The siRNAs seem to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells. These observations may open a path toward the use of siRNAs as a reverse genetic and therapeutic tool in mammalian cells.

Animals↗
PubMed2003

A calmodulin-binding protein from Arabidopsis has an essential role in pollen germination.

Calmodulin (CaM), a ubiquitous multifunctional calcium sensor in all eukaryotes, mediates calcium action by regulating the activity/function of many unrelated proteins. Although calcium and CaM are known to play a crucial role in pollen germination and pollen tube growth, the proteins that mediate their action have not been identified. We isolated three closely related CaM-binding proteins (NPG1, NPGR1, and NPGR2) from Arabidopsis. NPG1 (No Pollen Germination1) is expressed only in pollen, whereas the NPG-related proteins (NPGR1 and NPGR2) are expressed in pollen and other tissues. The bacterially expressed NPG1 bound three isoforms of Arabidopsis CaM in a calcium-dependent manner. To analyze the function of NPG1, we performed a reverse genetics screen and isolated a mutant in which NPG1 is disrupted by a T-DNA insertion. Segregation and molecular analyses of the NPG1 knockout mutant and a cross with a male sterile mutant indicate that the mutated NPG1 is not transmitted through the male gametophyte. Expression of NPG1 in the knockout mutant complemented the mutant phenotype. Analysis of pollen development in the knockout mutant by light microscopy showed normal pollen development. Pollen from NPG1 mutant in the quartet background has confirmed that NPG1 is dispensable for pollen development. However, germination studies with pollen from the mutant in the quartet background indicate that pollen carrying a mutant allele does not germinate. Our genetic, histological, and pollen germination studies with the knockout mutant line indicate that NPG1 is not necessary for microsporogenesis and gametogenesis but is essential for pollen germination.

Alleles↗
PubMed2003

The small RING finger protein Z drives arenavirus budding: implications for antiviral strategies.

By using a reverse genetics system that is based on the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), we have identified the arenavirus small RING finger Z protein as the main driving force of virus budding. Both LCMV and Lassa fever virus (LFV) Z proteins exhibited self-budding activity, and both substituted efficiently for the late domain that is present in the Gag protein of Rous sarcoma virus. LCMV and LFV Z proteins contain proline-rich motifs that are characteristic of late domains. Mutations in the PPPY motif of LCMV Z severely impaired the formation of virus-like particles. LFV Z contains two different proline-rich motifs, PPPY and PTAP, which are separated by eight amino acids. Mutational analysis revealed that both motifs are required for efficient LFV Z-mediated budding. Both LCMV and LFV Z proteins recruited to the plasma membrane Tsg101, which is a component of the class E vacuolar protein sorting machinery that has been implicated in budding of HIV and Ebola virus. Targeting of Tsg101 by RNA interference caused a strong reduction in Z-mediated budding. These results indicate that Z is the arenavirus functional counterpart of the matrix proteins found in other negative strand enveloped RNA viruses. Moreover, members of the vacuolar protein sorting pathway appear to play an important role in arena-virus budding. These findings open possibilities for antiviral strategies to combat LFV and other hemorrhagic fever arenaviruses.

Amino Acid Sequence↗
PubMed2002

An Arabidopsis histidine kinase is essential for megagametogenesis.

Cytokinin-Independent 1 (CKI1) belongs to a group of putative plant histidine kinases whose members do not appear to act as ethylene receptors. The deduced protein structure, combined with the observation that Arabidopsis callus cultures overexpressing CKI1 exhibit a "cytokinin-independent" cell division and greening phenotype, led to the hypothesis that CKI1 is involved in cytokinin signaling, perhaps acting as a cytokinin receptor. To test the function of CKI1, we used a reverse-genetic approach to identify plants carrying T-DNA insertions in CKI1. Two independent alleles were identified, which produce the same developmental phenotype. Analyses of populations segregating for the cki1-5 or cki1-6 T-DNA insertion alleles failed to reveal any homozygous cki1 plants, indicating that the homozygous mutant condition was lethal. Based on segregation distortion, transmission studies, a microscopy-based examination of developing female gametophytes, and mRNA expression data, we suggest that CKI1 function is required for megagametophyte development. Our work with CKI1 mutants indicates that signal transduction by means of a HisAsp phosphorelay system may play an important and previously unsuspected role in female gametophyte development in Arabidopsis.

Alleles↗
PubMed1987

Selective elimination of mRNAs in vivo: complementary oligodeoxynucleotides promote RNA degradation by an RNase H-like activity.

Oligodeoxynucleotides lead to translation arrest of complementary mRNAs in the wheat germ translation system by a degradation of the mRNA. In an attempt to develop an effective reverse genetic approach in vivo, we demonstrate that injection of short (15- to 30-nucleotide) oligonucleotides into Xenopus oocytes leads to complete degradation of both injected and endogenous mRNAs by means of an RNase H-like activity.

Animals↗
PubMed1989

Targeted gene mutations in Drosophila.

A cloned gene can be of interest because of its expression in a particular tissue or at a certain developmental stage, or because of homology to an interesting gene from another organism. In Drosophila its location in the genome is readily determined by in situ hybridization to the banded larval salivary gland polytene chromosomes, but it is more difficult to isolate mutations that may reveal its function. This paper describes a general method for detecting transposable element insertions into the gene in question. This "reverse genetics" then offers the possibility of observing a consequent mutant phenotype, providing a key to the normal function of the gene. The sensitivity of the polymerase chain reaction makes it possible to detect the occurrence of a single appropriate P-element transposon insertion among a population of mutagenized flies. This is accomplished by the use of oligonucleotide primers--one a sequence from within the cloned gene and the other homologous to the terminal sequence of the P-element DNA--to prime synthesis into the DNA flanking an insertion site. A segment of DNA, bounded by the two primers, will be a target for amplification only in a fly in which a P-element has inserted within about 2 kilobases of the gene primer. This technique has been used to detect P-element insertions near a gene expressed in the Drosophila compound eye. Potential problems with the technique and possible refinements in the screen are discussed. In principle, it could be utilized to detect insertion of a foreign element into any gene for which at least a partial sequence is known and could be extended to other organisms.

Animals↗
PubMed1991

Splicing-defective mutants of the yeast mitochondrial COXI gene can be corrected by transformation with a hybrid maturase gene.

We have developed a recombinant vector, termed pMIT, for transient expression of genes delivered to yeast mitochondria by biolistic transformation. Using that vector, we introduced a hybrid RNA maturase (splicing) gene into mitochondria of rho 0 petite cells and showed the gene to be functional in crosses. The hybrid maturase is an in-frame fusion between the N-terminal half of the maturase encoded by intron 1 of the COXI (cytochrome oxidase) gene and the C-terminal half of a similar maturase encoded by COXI intron 2. pMIT transformants can provide a functional maturase in crosses to restore respiration and COXI polypeptide synthesis to a respiratory-deficient strain defective in the synthesis of a maturase encoded by COXI intron 1; the transformant will also restore respiration to two splicing-defective cis mutants of COXI introns 1 and 3. We detect a 68-kDa polypeptide comparable in abundance to other major mitochondrial translation products as a likely product of the hybrid maturase gene. Transformants containing an internal 218-amino acid deletion mutation of the hybrid maturase gene no longer express a functional maturase in crosses and produce the expected shortened polypeptide of approximately 40 kDa; however, those transformants still restore respiration to the COXI cis mutants. These studies show the utility of the pMIT transformation system for the expression and reverse genetic analysis of yeast mitochondrial genes.

Cloning, Molecular↗
PubMed1991

The vinyl chloride DNA derivative N2,3-ethenoguanine produces G----A transitions in Escherichia coli.

Vinyl chloride is a known human and rodent carcinogen that forms several cyclic base derivatives in DNA. The mutagenic potential of these derivatives has been examined in vitro but not in vivo. One of these derivatives, N2,3-ethenoguanine (epsilon G), is known to base pair with both cytosine and thymine during in vitro DNA synthesis, which would result in G----A transitions. To determine the base pairing specificity of this labile guanine derivative in Escherichia coli, we have developed a genetic reversion assay for guanine derivatives. The assay utilizes DNA polymerase-mediated analogue insertion into a bacteriophage vector, M13G*1, which detects all single-base substitutions at position 141 of the lacZ alpha gene by change in plaque color. After the insertion of a single epsilon G opposite the template cytosine at position 141 by use of epsilon dGTP and DNA polymerase and further extension with all four normal dNTPs, the DNA was transfected into E. coli. Transfection of M13G*1 containing epsilon G at the target site yielded 135 mutants among 26,500 plaques, 134 of which represented G----A transitions. The uncorrected mutation frequency was 0.5%, as compared with the control value, approximately 0.02%; when corrected for epsilon G content and penetrance, the calculated mutagenic potential of epsilon G (mutations/analogue) was about 13%. We thus conclude that epsilon G specifically induces G----A transitions during DNA replication in E. coli. The M13G*1 assay may permit the testing of other labile guanine derivatives not otherwise amenable to mutagenesis studies.

Adenine↗
PubMed1992

High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates.

This report describes a method for growing both bloodstream- and procyclic-form Trypanosoma brucei as colonies on agarose plates. Procyclic colonies, which took 2 weeks to develop, grew with approximately 17% plating efficiency on SDM-79/0.65% agarose supplemented with 20% (vol/vol) conditioned medium. Bloodstream forms were adapted to in vitro growth in liquid HMI-9 medium and then spread on HMI-9/0.65% agarose plates, where they grew to visible colonies in 3-5 days. Plating efficiencies were from 3 to 80%, depending upon the trypanosome variant and experiment. Colonies were proven to be the result of growth from a single cell and contained approximately 10(6) cells at maturity. Colonies were transferred to filters and probed for multicopy and single-copy genes. Potential uses of this method in conjunction with classical and reverse genetic approaches to studying trypanosomes are discussed.

Animals↗
PubMed1992

Intrahypothalamic injection of a cell line secreting gonadotropin-releasing hormone results in cellular differentiation and reversal of hypogonadism in mutant mice.

GT1 is an immortalized cell line that synthesizes and secretes the neurohormone gonadotropin-releasing hormone (GnRH). We have placed these cells into the brains of adult mutant hypogonadal (hpg) mice, which lack a functional GnRH gene, to determine whether such cells could differentiate in situ and support gonadal development. Immunocytochemical detection of GnRH revealed that these cells migrated widely in the central nervous system and elaborated axonal processes which on rare occasion projected to the normal target, the median eminence. Using a battery of antibodies, we demonstrated that these cells could cleave the GnRH precursor and that the amidated decapeptide as well as other cleavage products were present. The presence of biologically active material and its appropriate secretion were further documented by gonadal growth in both males and females. The morphological differentiation of the GT1 cells correlated with the density of cells injected. Those remaining within the injection site and/or forming a tumor retained a simple, rounded or fibroblastic appearance. Those cells that migrated into the host away from such tumors assumed the simple fusiform shape of normal GnRH neurons with dendrites extending from one or both poles. When cell density was drastically reduced a much more complex dendritic arbor was elaborated. These data suggest that such cell lines can be useful in reversing genetic defects and in studying such processes as GnRH neuronal migration, axonal targeting, and cytological differentiation.

Animals↗
PubMed1993

Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank.

To understand how genotype determines the phenotype of the animal Caenorhabditis elegans, one ideally needs to know the complete sequence of the genome and the contribution of genes to phenotype, which requires an efficient strategy for reverse genetics. We here report that the Tc1 transposon induces frequent deletions of flanking DNA, apparently resulting from Tc1 excision followed by imprecise DNA repair. We use this to inactivate genes in two steps. (i) We established a frozen library of 5000 nematode lines mutagenized by Tc1 insertion, from which insertion mutants of genes of interest can be recovered. Their address within the library is determined by PCR. (ii) Animals are then screened, again by PCR, to detect derivatives in which Tc1 and 1000-2000 base pairs of flanking DNA are deleted, and thus a gene of interest is inactivated. We have thus far isolated Tc1 insertions in 16 different genes and obtained deletion derivatives of 6 of those.

Alleles↗
PubMed1994

Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli.

The termination of protein synthesis in bacteria requires two codon-specific polypeptide release factors, RF-1 and RF-2. A third factor, RF-3, which stimulates the RF-1 and RF-2 activities, was originally identified in Escherichia coli, but it has received little attention since the 1970s. To search for the gene encoding RF-3, we selected nonsense-suppressor mutations by random insertion mutagenesis on the assumption that a loss of function of RF-3 would lead to misreading of stop signals. One of these mutations, named tos-1 (for transposon-induced opal suppressor), mapped to the 99.2 min region on the E. coli chromosome and suppressed all three stop codons. Complementation studies and analyses of the DNA and protein sequences revealed that the tos gene encodes a 59,442-Da protein, with sequence homology to elongation factor EF-G, including G-domain motifs, and that the tos-1 insertion eliminated the C-terminal one-fifth of the protein. Extracts containing the overproduced Tos protein markedly increased the formation of ribosomal termination complexes and stimulated the RF-1 or RF-2 activity in the codon-dependent in vitro termination assay. The stimulation was significantly reduced by GTP, GDP, and the beta,gamma-methylene analog of GTP, but not by GMP. These results fit perfectly with those described in the original publications on RF-3, and the tos gene has therefore been designated prfC. A completely null prfC mutation made by reverse genetics affected the cell growth under the limited set of physiological and strain conditions.

Amino Acid Sequence↗
PubMed1994

Bloom syndrome: an analysis of consanguineous families assigns the locus mutated to chromosome band 15q26.1.

By the principle of identity by descent, parental consanguinity in individuals with rare recessively transmitted disorders dictates homozygosity not just at the mutated disease-associated locus but also at sequences that flank that locus closely. In 25 of 26 individuals with Bloom syndrome examined whose parents were related, a polymorphic tetranucleotide repeat in an intron of the protooncogene FES was homozygous, far more often than expected (P < 0.0001 by chi 2). Therefore, BLM, the gene that when mutated gives rise to Bloom syndrome, is tightly linked to FES, a gene whose chromosome position is known to be 15q26.1. This successful approach to the assignment of the Bloom syndrome locus to one short segment of the human genome simultaneously (i) demonstrates the power of homozygosity mapping and (ii) becomes the first step in a "reverse" genetics definition of the primary defect in Bloom syndrome.

Alleles↗
PubMed1994

Nature screen: an efficient method for screening natural populations of Drosophila for targeted P-element insertions.

The efficiency of molecular techniques is making it increasingly necessary to rely on reverse genetics to understand the function of genes. Tissue-specific libraries allow one to identify numerous genes that can be cloned, sequenced, and mapped and whose temporal and tissue-specific pattern of expression are well characterized but whose function remains unknown. In such cases, it is desirable to generate targeted mutations to examine the phenotype of loss-of-function lesions. Here we describe a method for identifying naturally occurring variants of Drosophila melanogaster with specific genes tagged by a nearby P element. Imprecise P-element excision can then be used to generate a series of small deletions in or near the gene. In the method described here, large numbers of wild-caught males were crossed to balancer females, and inserts were identified in pooled samples by the polymerase chain reaction with one primer from each target gene and one primer from the P-element terminal repeat. We present the calculations for the probability of successfully tagging a gene and show that it is greatly improved by simultaneously screening inserts into several genes. If a large natural population is available, a nature screen is faster and easier than inducing P-element transposition in the laboratory, but the resulting lines, being genetically heterogeneous, may require more subsequent work to isolate. Using this method to screen the genomes of approximately 10,400 males, we found P-element inserts in close proximity to 3 of 10 genes that were screened.

Animals↗