Solid-phase sequence scanning for drug resistance detection in tuberculosis.

DNA chip arrays hold considerable promise for diagnostic sequencing of polymerase chain reaction (PCR) products. To date, however, arrays have been relatively expensive, complex to use and difficult to interpret, preventing their adaptation to the clinical lab. A moderate density array method has been developed that enables efficient, easy-to-interpret and robust solid-phase PCR product sequencing. Here, the results of Mycobacterium tuberculosis rifampin resistance mutation detection by primer-extension-based sequence scanning of the rpo B gene of M. tuberculosis are presented. Rifampin resistant clinical isolates were identified in as little as 1 h post PCR amplification with visual results detection.

Nested genetic bit analysis (N-GBA) for mutation detection in the p53 tumor suppressor gene.

There is a growing and significant demand for reliable, simple and sensitive methods for repeated scanning of a given gene or gene fragment for detection and characterization of mutations. Solid-phase sequencing by single base primer extension of nested GBATM primers on miniaturized DNA arrays can be used to effectively scan targeted sequences for missense, insertion and deletion mutations. This paper describes the use of N-GBA arrays designed to scan the sequence of a 33 base region of exon 8 of the p53 gene (codons 272-282) encompassing a hot spot for mutations associated with the development of cancer. Synthetic DNA templates containing various missense, insertion and deletion mutations, as well as DNA prepared from pancreatic and biliary tumor cells, were genotyped using the exon 8 arrays.

Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.

A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.

Specific block of calcium channel expression by a fragment of dihydropyridine receptor cDNA.

Although the structure of rabbit skeletal muscle dihydropyridine (DHP) receptor, deduced from cDNA sequence, indicates that this protein is the channel-forming subunit of voltage-dependent calcium channel (VDCC), no functional proof for this prediction has been presented. Two DNA oligonucleotides complementary to DHP-receptor RNA sequences coding for putative membrane-spanning regions of the DHP receptor specifically suppress the expression of the DHP-sensitive VDCC from rabbit and rat heart in Xenopus oocytes. However, these oligonucleotides do not suppress the expression of the DHP-insensitive VDCC and of voltage-dependent sodium and potassium channels. Thus, the gene for DHP receptor of rabbit skeletal muscle is closely related, or identical to, a gene expressed in heart that encodes a component of the DHP-sensitive VDCC. The DHP-sensitive and DHP-insensitive VDCCs are distinct molecular entities.

Inhibitor of protein synthesis blocks long-term behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia.

To study the effects of protein synthesis inhibition on long-term sensitization of the gill- and siphon-withdrawal reflex of Aplysia, we have developed an isolated reflex preparation in which we could expose the inhibitor to only that part of the central nervous system involved in mediating the reflex and not to the other parts of the animal's central nervous system, thus minimizing the possible systemic side effects. We have found that long-term sensitization can be obtained in the isolated gill reflex, and that this long-term process, but not the short-term process, is blocked selectively by anisomycin, a reversible inhibitor of protein synthesis. Moreover, to obtain this blockade of long-term sensitization, this drug need only be applied during the training procedure.

Blockade of ion channel expression in Xenopus oocytes with complementary DNA probes to Na+ and K+ channel mRNAs.

Ionic currents were recorded from Xenopus oocytes injected with RNA isolated from chick or mouse brain. Three currents were studied: a rapid tetrodotoxin-sensitive Na+ current (Ina), an early outward K+ current sensitive to 4-aminopyridine (IA), and an inward current activated by the excitatory amino acid receptor agonist kainate. Oligonucleotides (60-80 bases long) complementary to rat brain Na+ channel sequences were prehybridized to chick brain RNA. These DNA sequences, upon injection into oocytes, specifically inhibited expression of INa relative to IA and the kainate-induced current in a dose-dependent manner. By contrast, prehybridization of oligonucleotides complementary to sequences either from the Drosophila Shaker locus (which codes for an early K+ current in Drosophila muscle) or from a homologous clone from mouse brain did not block the expression of the early outward K+ current induced in the oocytes by mRNA from chick or mouse brain. This method provides a convenient means for testing the functional role of cloned DNA species.

A quantitative analysis of 2-D gels identifies proteins in which labeling is increased following long-term sensitization in Aplysia.

Long-term memory for sensitization of the gill- and siphon-withdrawal reflex in Aplysia, produced by 4 days of training, is associated with increased synaptic efficacy of the connection between the sensory and motor neurons. This training is also accompanied by neuronal growth; there is an increase in the number of synaptic varicosities per sensory neuron and in the number of active zones. Such structural changes may be due to changes in the rates of synthesis of certain proteins. We have searched for proteins in which the rates of [35S]methionine labeling are altered during the maintenance phase of long-term memory for sensitization by using computer-assisted quantitative 2-D gel analysis. This method has allowed us to detect 4 proteins in which labeling is altered after 4 days of sensitization training.

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.

A critical period for macromolecular synthesis in long-term heterosynaptic facilitation in Aplysia.

Both long-term and short-term sensitization of the gill and siphon withdrawal reflex in Aplysia involve facilitation of the monosynaptic connections between the sensory and motor neurons. To analyze the relationship between these two forms of synaptic facilitation at the cellular and molecular level, this monosynaptic sensorimotor component of the gill-withdrawal reflex of Aplysia can be reconstituted in dissociated cell culture. Whereas one brief application of 1 microM serotonin produced short-term facilitation in the sensorimotor connection that lasted minutes, five applications over 1.5 hours resulted in long-term facilitation that lasted more than 24 hours. Inhibitors of protein synthesis or RNA synthesis selectively blocked long-term facilitation, but not short-term facilitation, indicating that long-term facilitation requires the expression of gene products not essential for short-term facilitation. Moreover, the inhibitors only blocked long-term facilitation when given during the serotonin applications; the inhibitors did not block the facilitation when given either before or after serotonin application. These results parallel those for behavioral performance in vertebrates and indicate that the critical time window characteristic of the requirement for macromolecular synthesis in long-term heterosynaptic facilitation is not a property of complex circuitry, but an intrinsic characteristic of specific nerve cells and synaptic connections involved in the long-term storage of information.

Some principles emerging from the study of short- and long-term memory.

Recent studies indicate that in invertebrates short-term memory for various forms of learning involves covalent modifications of pre-existing proteins. By contrast, long-term memory utilizes genes and proteins not required for short-term memory.

The long and the short of long-term memory--a molecular framework.

A single learning event initiates several memory processes with different time courses of retention. While short term memory involves covalent modification of pre-existing proteins, the finding that long-term memory requires the expression, during learning, of additional genes, makes it possible to analyse in molecular terms the induction and retention of long-term memory.

Cell and molecular analysis of long-term sensitization in Aplysia.

We have found that one cellular locus for the storage of the memory underlying short-term sensitization of the gill and siphon withdrawal reflex in Aplysia is the set of monosynaptic connections between the siphon sensory cells and the gill and siphon motor neurons. These connections also participate in the storage of memory underlying long-term sensitization. In animals that have undergone long-term sensitization, the amplitudes of the monosynaptic connections are significantly larger (2.2x) than the ones in control animals. To study the mechanisms of onset and retention of long-term synaptic facilitation that underly long-term sensitization and the role of protein synthesis in long-term memory, we have developed two types of reduced preparations: the intact reflex isolated from the remainder of the animal, and a dissociated cell culture system in which the monosynaptic component (sensory neurons and motor neurons) of the neuronal circuit mediating the withdrawal reflex is reconstituted. We found that protein synthesis inhibitors, such as anisomycin or emetine, and RNA synthesis inhibitors, such as actinomycin D or alpha-amanitin, blocked long-term facilitation without interfering with short-term facilitation. These results suggest that the acquisition of long-term memory may require the expression of genes and the synthesis of proteins not needed for short-term memory.

Expression of tobacco mosaic virus coat protein by a cauliflower mosaic virus promoter in plants transformed by Agrobacterium.

The 35S transcript promoter of cauliflower mosaic virus directs the synthesis of an abundant RNA in infected leaf tissue that is both a template for reverse transcription during virus replication and also a polycistronic mRNA. The 35S promoter was isolated and linked to a cDNA of the 3' end of tobacco mosaic virus that encodes the coat protein of this single-stranded RNA virus. After transformation of this gene construction into tobacco plants using a disarmed Agrobacterium binary vector, viral coat protein synthesis was detected in transformed leaf tissue. A chimaeric 35S coat protein RNA was detected in transformed callus.

Methods for cDNA cloning and sequencing tobacco mosaic virus RNA.

DNA complementary to tobacco mosaic virus (TMV) RNA (cDNA) was prepared by priming reverse transcription with synthetic oligonucleotides. The cDNAs terminated prematurely at many specific sites and no transcripts longer than about 2000 nucleotides were obtained. However, the entire 6395 nucleotide long TMV RNA could be copied into cDNA by specific priming with a series of 13 to 17 residue long oligonucleotides or by non-specific priming with short, 4 to 7 residue, oligonucleotides. A number of different priming methods were used to convert the cDNA into double-stranded DNA (dsDNA). The double-stranded cDNA was recovered by shotgun cloning into M13 and analysed by sequencing. The frequency at which cDNA clones were recovered has been used to compare various cDNA cloning strategies.

Striking similarities in amino acid sequence among nonstructural proteins encoded by RNA viruses that have dissimilar genomic organization.

The plant viruses alfalfa mosaic virus (AMV) and brome mosaic virus (BMV) each divide their genetic information among three RNAs while tobacco mosaic virus (TMV) contains a single genomic RNA. Amino acid sequence comparisons suggest that the single proteins encoded by AMV RNA 1 and BMV RNA 1 and by AMV RNA 2 and BMV RNA 2 are related to the NH2-terminal two-thirds and the COOH-terminal one-third, respectively, of the largest protein encoded by TMV. Separating these two domains in the TMV RNA sequence is an amber termination codon, whose partial suppression allows translation of the downstream domain. Many of the residues that the TMV read-through domain and the segmented plant viruses have in common are also conserved in a read-through domain found in the nonstructural polyprotein of the animal alphaviruses Sindbis and Middelburg. We suggest that, despite substantial differences in gene organization and expression, all of these viruses use related proteins for common functions in RNA replication. Reassortment of functional modules of coding and regulatory sequence from preexisting viral or cellular sources, perhaps via RNA recombination, may be an important mechanism in RNA virus evolution.

Sensitive detection of RNA using strand-specific M13 probes.

We have extended the method of Hu and Messing (Gene 17 (1982) 271-277) to prepare highly radioactive M13 probes suitable for use in RNA-DNA hybridization experiments. Single strands of M13 DNA carrying cloned sequences are rendered partially double-stranded by primed synthesis using a synthetic oligonucleotide primer complementary to a region 5' to the cloning site. The newly synthesized radioactive complementary strand is then covalently cross-linked to the M13 phage DNA by UV irradiation in the presence of 4,5,8-trimethylpsoralen (trioxsalen). Since the cross-linked probe is stable to heat denaturation, and the region of cloned sequence is kept single-stranded, these complexes may be used as strand-specific hybridization probes to detect RNA sequences under conditions which would denature DNA-DNA duplexes.