Conrad Lichtenstein - Academia.edu (original) (raw)
Papers by Conrad Lichtenstein
Archives of Virology, 2003
Cotton, the major cash crop in Pakistan, suffers 30% losses to cotton leaf curl disease, caused b... more Cotton, the major cash crop in Pakistan, suffers 30% losses to cotton leaf curl disease, caused by the geminivirus, cotton leaf curl virus DNA A, plus a satellite component, DNA β responsible for symptom development with plants failing to produce cotton bolls. We constructed transgenic tobacco expressing sense and antisense RNAs representing: [i] the 5 half of the viral DNA replication gene, AC1, [ii] the 3 half of AC1, [iii] two overlapping genes, AC2, a transcription activator, and AC3, a replication enhancer. In contrast to controls, 25% of 72 transgenic tobacco lines tested showed heritable resistance [T 1 − T 3 generations]: symptom-free and no replication of DNA A or DNA β even after 120 days of continuous exposure to viruliferous whiteflies. As geminiviral and transgene RNAs are not detected in resistant lines following infection, and selected uninfected resistant tobacco sense lines reveal double-stranded and small interfering RNAs, the most likely mechanism is via post-transcriptional gene silencing.
Archives Of Phytopathology And Plant Protection, 2001
A system combining antisense RNA and RNAseIII‐binding activity was designed. Two antisense RNA ve... more A system combining antisense RNA and RNAseIII‐binding activity was designed. Two antisense RNA vehicles, 7SL RNA and tRNA genes containing the coat protein leader domain (2245–2316) and “zinc finger”; domain (3325–3411) of PVS, respectively were prepared. Both, antisense RNA vehicles were shown to be efficiently transcribed by polymerase III using in vitro assay. It was shown by specific RT PCR,
1: Nucleic acid structures. 2: Evaluation of antisense effects. 3: Selecting, preparing, and hand... more 1: Nucleic acid structures. 2: Evaluation of antisense effects. 3: Selecting, preparing, and handling antisense oligodeoxyribonucleotides. 4: In vitro RNAs. 5: Catalytic antisense RNA based on hammerhead ribozymes. 6: 2-5 A-Antisense chimaeras for targeted degradation of RNA. 7: In vitro applications of antisense RNA and DNA. 8: Antisense applications in Dictyostelium: a lower eukaryotic model system. 9: Applications of antisense technology in plants. 10: Antisense molecules and ribozymes: medical applications. 11: Non-antisense effects of oligodeoxynucleotides. 12: Antisense rescue. 13: Shotgun antisense mutants. 14: Detection of sense-antisense duplexes by structure-specific anti-RNA antibodies
SUMMARY Mutations in the terminal 8-bp (5'-T1G2T3G4GSG6CVGS-3 ') of the inverted repeats ... more SUMMARY Mutations in the terminal 8-bp (5'-T1G2T3G4GSG6CVGS-3 ') of the inverted repeats of the bacterial transposon, Tn7, were analysed by measuring Tn7 transposition to the attachment site, attTn7. The mutation, C 2, present at either end of Tn7 reduces transposition only threefold, but in the double mutant, with C 2 at both ends of Tn7, no transposition is detected. C 6 mutations have no effect on transposition frequency. Replacement with 5'-A3C4GSC6GVCS-3' at the right end of Tn7 apparently abolishes transposition; yet in the double mutant, where the inverted repeats are restored by substituting this sequence at both ends of Tn7, transposition is partially rescued. This suggests that the mechanism of Tn7 transposition requires communication between the two ends. Tn7 transposition has always been seen to generate a 5-bp target duplication. This is presumed to result from a staggered cut, plus repair synthesis during transposition. We found that two of our right-en...
Chromosoma, 2000
Phylogenetic schemes based on changing DNA sequence have made a major impact on our understanding... more Phylogenetic schemes based on changing DNA sequence have made a major impact on our understanding of evolutionary relationships and significantly built on knowledge gained by morphological and anatomical studies. Here we present another approach to phylogeny, using fluorescent in situ hybridisation. The phylogenetic scheme presented is likely to be robust since it is derived from the chromosomal distribution of ten repetitive sequences with different functions and evolutionary constraints [GRS, HRS60, NTRS, the Arabidopsis-type telomere repeat (TTTAGGG)n, 18S-5.8S-26S ribosomal DNA (rDNA), 5S rDNA, and four classes of geminiviral-related DNA (GRD)]. The basic karyotypes of all the plant species investigated Nicotiana tomentosiformis, N. kawakamii, N. tomentosa, N. otophora, N. setchellii, N. glutinosa (all section Tomentosae), and N. tabacum (tobacco, section Genuinae) are similar (x=12) but the distribution of genic and non-genic repeats is quite variable, making the karyotypes distinct. We found sequence dispersal, and locus gain, amplification and loss, all within the regular framework of the basic genomic structure. We predict that the GRD classes of sequence integrated into an ancestral genome only once in the evolution of section Tomentosae and thereafter spread by vertical transmission and speciation into four species. Since GRD is similar to a transgenic construct that was inserted into the N. tabacum genome, its fate over evolutionary time is interesting in the context of the debate on genetically modified organisms and the escape of genes into the wild. Nicotiana tabacum is thought to be an allotetraploid between presumed progenitors of N. sylvestris (maternal, S-genome donor) and a member of section Tomentosae (T-genome donor). Of section Tomentosae, N. tomentosiformis has the most similar genome to the T genome of tobacco and is therefore the most likely paternal genome donor. It is known for N. tabacum that gene conversion has converted most 18S-5.8S-26S rDNA units of N. sylvestris origin into units of an N. tomentosiformis type. Clearly if such a phenomenon were widespread across the genome, genomic in situ hybridisation (GISH) to distinguish the S and T genomes would probably not work since conversion would tend to homogenise the genomes. The fact that GISH does work suggests a limited role for gene conversion in the evolution of N. tabacum.
Chromosoma, 2000
We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nico... more We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nicotiana sylvestris (2n=2x=24) and N. tomentosiformis (2n=2x=24) and compared these with patterns in N. tabacum (tobacco, 2n=4x=48). We also examined a longestablished N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosinesensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N. sylvestris type (from ca. 75% based on the sum of the rDNA copy numbers in the parents). Since the active genes are likely to be of an N. tomentosiformis type, the N. sylvestris type units are presumably contained within inactive loci (i.e. on chromosome S12). Nicotiana sylvestris has approximately three times as much rDNA as the other two species, resulting in much condensed rDNA at interphase. This species also has three classes of IGS, indicating gene conversion has not homogenised repeat length in this species. The results suggest that methylation and/or DNA condensation has reduced or prevented gene conversion from occurring at inactive genes at rDNA loci. Alternatively, active undermethylated units may be vulnerable to gene conversion, perhaps because they are decondensed and located in close proximity within the nucleolus at interphase. In TBY-2, restriction enzymes showed hybridisation patterns that were similar to, but different from, those of N. tabacum. In addition, TBY-2 has elevated rDNA copy number and variable numbers of rDNA loci, all indicating rDNA evolution in culture.
Gene, 1995
Mutations in the terminal 8-bp (5'-T1G2T3G4G5G6C7G8-3') of the inverted repeats o... more Mutations in the terminal 8-bp (5'-T1G2T3G4G5G6C7G8-3') of the inverted repeats of the bacterial transposon, Tn7, were analysed by measuring Tn7 transposition to the attachment site, attTn7. The mutation, C2, present at either end of Tn7 reduces transposition only threefold, but in the double mutant, with C2 at both ends of Tn7, no transposition is detected. C6 mutations have no effect on transposition frequency. Replacement with 5'-A3C4G5C6G7C8-3' at the right end of Tn7 apparently abolishes transposition; yet in the double mutant, where the inverted repeats are restored by substituting this sequence at both ends of Tn7, transposition is partially rescued. This suggests that the mechanism of Tn7 transposition requires communication between the two ends. Tn7 transposition has always been seen to generate a 5-bp target duplication. This is presumed to result from a staggered cut, plus repair synthesis during transposition. We found that two of our right-end mutants, C2 and C6, sometimes yielded a 6-bp target duplication. This observation implies that cleavage of the target site might also involve interaction with the donor ends which, when mutant, relax the specificity for target-site cleavage.
EL DOCTOR CONRAD LICHTENSTEIN, CIENTIFICO DEL CENTRO DE BIOTECNOLOGIA DEL IMPERIAL COLLEGE DEL LO... more EL DOCTOR CONRAD LICHTENSTEIN, CIENTIFICO DEL CENTRO DE BIOTECNOLOGIA DEL IMPERIAL COLLEGE DEL LONDRES, HA CREADO Y COMENZADO A PROBAR UN NUEVO METODO REVOLUCIONARIO DE PROTECCION DE LOS CULTIVOS CONTRA ENFERMEDADES VIRALES, MEDIANTE EL EMPLEO DE TECNICAS DE MANIPULACION GENETICA.
The Plant Cell Online, 1992
Homologous recombination has been extensively studied in bacteria, yeast, and more recently in an... more Homologous recombination has been extensively studied in bacteria, yeast, and more recently in animal cells, but little is known about this process in plants. We present here an analysis of meiotic and somatic chromosomal recombination between closely linked ...
Proceedings of the …, 1996
Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and anim... more Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and animal systems, has surprisingly not been reported for plants. We have discovered geminivirus-related DNA (GRD) sequences, in the form of distinct sets of multiple direct repeats comprising three related repeat classes, situated in a unique locus in the Nicotiana tabacum (tobacco) nuclear genome. The organization of these sequences is similar or identical in eight different tobacco cultivars we have examined. DNA sequence analysis reveals that each repeat has sequences most resembling those of the New World geminiviral DNA replication origin plus the adjacent AL1 gene, encoding the viral replication protein. We believe these GRD sequences originated quite recently in Nicotiana evolution through integration of geminiviral DNA by some combination of the processes of illegitimate recombination, amplification, deletions, and rearrangements. These events must have occurred in plant tissue that was subsequently able to contribute to meristematic tissue yielding gametes. GRD may have been retained in tobacco by selection or by random fixation in a small evolving population. Although we cannot detect transcription of these sequences, this does not exclude the possibility that they may originally have been expressed.
Trends in Biotechnology, 1992
The use of antisense RNA to manipulate plant metabolism has already beell shown to be successful ... more The use of antisense RNA to manipulate plant metabolism has already beell shown to be successful in altering pigment production in flowers and maniptrlating the ripening and softening processes in fruit. The major losses in crop yields due to viral pathogens hFve led to kvestigations of many strategies for developing *virusresistant plants. By inhibiting viral replication, the use of antisense may permit the engineering of such resistanI plants.
Un metodo de estimacion del numero de moleculas de polinucleotidos de partida secuenciadas a part... more Un metodo de estimacion del numero de moleculas de polinucleotidos de partida secuenciadas a partir de multiples muestras, comprendiendo el metodo: unir un adaptador a las moleculas de polinucleotidos de partida de multiples muestras diferentes, en donde el adaptador para cada muestra comprende: una marca de un unico identificador multiplex (MID) especifico para la muestra, haciendo posible la marca del identificador multiplex la identificacion de la muestra a partir de la cual se deriva un polinucleotido; y una region de bases degeneradas (DBR) que comprende al menos una base de nucleotidos seleccionada de: R, Y, S, W, K, M, B, D, H, V, N, y las versiones modificadas de las mismas, siendo la degeneracion de las DBR de tal modo que es probable que cada polinucleotido individual tenga una DBR diferente; reunir las multiples muestras unidas a adaptadores diferentes para generar una muestra conjunta; amplificar los polinucleotidos unidos al adaptador en la muestra conjunta; secuenciar ...
Des aspects de la presente invention concernent des procedes et des compositions pour la determin... more Des aspects de la presente invention concernent des procedes et des compositions pour la determination du nombre de molecules individuelles de polynucleotide issues de la meme region genomique du meme echantillon d'origine qui ont ete sequencees dans une configuration particuliere ou un procede particulier d'analyse de sequence. Dans ces aspects de l'invention, une region de base degeneree (DBR) est fixee aux molecules initiales de polynucleotide qui sont ulterieurement sequencees (par exemple, apres que certaines etapes du procede sont realisees, par exemple, l'amplification et/ou l'enrichissement). Le nombre de sequences DBR differentes presentes dans un cycle de sequencage peut etre utilise pour determiner/estimer le nombre de polynucleotides initiaux differents qui ont ete sequences. Les DBR peuvent etre utilisees pour augmenter de nombreuses applications differentes d'analyse de sequences d'acides nucleiques, permettant notamment des determinations d...
BMC Proceedings
We are developing novel methods to simultaneously analyze candidate genes or regions from multipl... more We are developing novel methods to simultaneously analyze candidate genes or regions from multiple samples in a single experiment rapidly and cost-effectively. One method, we call Reflex, provides an elegant approach to sample preparation for long-range PCR (LR-PCR) amplicons. Current methods use LR-PCR for target enrichment then use random fragmentation of each sample followed by ligation of DNA barcodes before sequencing: this approach is expensive and labour intensive.
Archives of Virology, 2003
Cotton, the major cash crop in Pakistan, suffers 30% losses to cotton leaf curl disease, caused b... more Cotton, the major cash crop in Pakistan, suffers 30% losses to cotton leaf curl disease, caused by the geminivirus, cotton leaf curl virus DNA A, plus a satellite component, DNA β responsible for symptom development with plants failing to produce cotton bolls. We constructed transgenic tobacco expressing sense and antisense RNAs representing: [i] the 5 half of the viral DNA replication gene, AC1, [ii] the 3 half of AC1, [iii] two overlapping genes, AC2, a transcription activator, and AC3, a replication enhancer. In contrast to controls, 25% of 72 transgenic tobacco lines tested showed heritable resistance [T 1 − T 3 generations]: symptom-free and no replication of DNA A or DNA β even after 120 days of continuous exposure to viruliferous whiteflies. As geminiviral and transgene RNAs are not detected in resistant lines following infection, and selected uninfected resistant tobacco sense lines reveal double-stranded and small interfering RNAs, the most likely mechanism is via post-transcriptional gene silencing.
Archives Of Phytopathology And Plant Protection, 2001
A system combining antisense RNA and RNAseIII‐binding activity was designed. Two antisense RNA ve... more A system combining antisense RNA and RNAseIII‐binding activity was designed. Two antisense RNA vehicles, 7SL RNA and tRNA genes containing the coat protein leader domain (2245–2316) and “zinc finger”; domain (3325–3411) of PVS, respectively were prepared. Both, antisense RNA vehicles were shown to be efficiently transcribed by polymerase III using in vitro assay. It was shown by specific RT PCR,
1: Nucleic acid structures. 2: Evaluation of antisense effects. 3: Selecting, preparing, and hand... more 1: Nucleic acid structures. 2: Evaluation of antisense effects. 3: Selecting, preparing, and handling antisense oligodeoxyribonucleotides. 4: In vitro RNAs. 5: Catalytic antisense RNA based on hammerhead ribozymes. 6: 2-5 A-Antisense chimaeras for targeted degradation of RNA. 7: In vitro applications of antisense RNA and DNA. 8: Antisense applications in Dictyostelium: a lower eukaryotic model system. 9: Applications of antisense technology in plants. 10: Antisense molecules and ribozymes: medical applications. 11: Non-antisense effects of oligodeoxynucleotides. 12: Antisense rescue. 13: Shotgun antisense mutants. 14: Detection of sense-antisense duplexes by structure-specific anti-RNA antibodies
SUMMARY Mutations in the terminal 8-bp (5'-T1G2T3G4GSG6CVGS-3 ') of the inverted repeats ... more SUMMARY Mutations in the terminal 8-bp (5'-T1G2T3G4GSG6CVGS-3 ') of the inverted repeats of the bacterial transposon, Tn7, were analysed by measuring Tn7 transposition to the attachment site, attTn7. The mutation, C 2, present at either end of Tn7 reduces transposition only threefold, but in the double mutant, with C 2 at both ends of Tn7, no transposition is detected. C 6 mutations have no effect on transposition frequency. Replacement with 5'-A3C4GSC6GVCS-3' at the right end of Tn7 apparently abolishes transposition; yet in the double mutant, where the inverted repeats are restored by substituting this sequence at both ends of Tn7, transposition is partially rescued. This suggests that the mechanism of Tn7 transposition requires communication between the two ends. Tn7 transposition has always been seen to generate a 5-bp target duplication. This is presumed to result from a staggered cut, plus repair synthesis during transposition. We found that two of our right-en...
Chromosoma, 2000
Phylogenetic schemes based on changing DNA sequence have made a major impact on our understanding... more Phylogenetic schemes based on changing DNA sequence have made a major impact on our understanding of evolutionary relationships and significantly built on knowledge gained by morphological and anatomical studies. Here we present another approach to phylogeny, using fluorescent in situ hybridisation. The phylogenetic scheme presented is likely to be robust since it is derived from the chromosomal distribution of ten repetitive sequences with different functions and evolutionary constraints [GRS, HRS60, NTRS, the Arabidopsis-type telomere repeat (TTTAGGG)n, 18S-5.8S-26S ribosomal DNA (rDNA), 5S rDNA, and four classes of geminiviral-related DNA (GRD)]. The basic karyotypes of all the plant species investigated Nicotiana tomentosiformis, N. kawakamii, N. tomentosa, N. otophora, N. setchellii, N. glutinosa (all section Tomentosae), and N. tabacum (tobacco, section Genuinae) are similar (x=12) but the distribution of genic and non-genic repeats is quite variable, making the karyotypes distinct. We found sequence dispersal, and locus gain, amplification and loss, all within the regular framework of the basic genomic structure. We predict that the GRD classes of sequence integrated into an ancestral genome only once in the evolution of section Tomentosae and thereafter spread by vertical transmission and speciation into four species. Since GRD is similar to a transgenic construct that was inserted into the N. tabacum genome, its fate over evolutionary time is interesting in the context of the debate on genetically modified organisms and the escape of genes into the wild. Nicotiana tabacum is thought to be an allotetraploid between presumed progenitors of N. sylvestris (maternal, S-genome donor) and a member of section Tomentosae (T-genome donor). Of section Tomentosae, N. tomentosiformis has the most similar genome to the T genome of tobacco and is therefore the most likely paternal genome donor. It is known for N. tabacum that gene conversion has converted most 18S-5.8S-26S rDNA units of N. sylvestris origin into units of an N. tomentosiformis type. Clearly if such a phenomenon were widespread across the genome, genomic in situ hybridisation (GISH) to distinguish the S and T genomes would probably not work since conversion would tend to homogenise the genomes. The fact that GISH does work suggests a limited role for gene conversion in the evolution of N. tabacum.
Chromosoma, 2000
We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nico... more We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nicotiana sylvestris (2n=2x=24) and N. tomentosiformis (2n=2x=24) and compared these with patterns in N. tabacum (tobacco, 2n=4x=48). We also examined a longestablished N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosinesensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N. sylvestris type (from ca. 75% based on the sum of the rDNA copy numbers in the parents). Since the active genes are likely to be of an N. tomentosiformis type, the N. sylvestris type units are presumably contained within inactive loci (i.e. on chromosome S12). Nicotiana sylvestris has approximately three times as much rDNA as the other two species, resulting in much condensed rDNA at interphase. This species also has three classes of IGS, indicating gene conversion has not homogenised repeat length in this species. The results suggest that methylation and/or DNA condensation has reduced or prevented gene conversion from occurring at inactive genes at rDNA loci. Alternatively, active undermethylated units may be vulnerable to gene conversion, perhaps because they are decondensed and located in close proximity within the nucleolus at interphase. In TBY-2, restriction enzymes showed hybridisation patterns that were similar to, but different from, those of N. tabacum. In addition, TBY-2 has elevated rDNA copy number and variable numbers of rDNA loci, all indicating rDNA evolution in culture.
Gene, 1995
Mutations in the terminal 8-bp (5'-T1G2T3G4G5G6C7G8-3') of the inverted repeats o... more Mutations in the terminal 8-bp (5'-T1G2T3G4G5G6C7G8-3') of the inverted repeats of the bacterial transposon, Tn7, were analysed by measuring Tn7 transposition to the attachment site, attTn7. The mutation, C2, present at either end of Tn7 reduces transposition only threefold, but in the double mutant, with C2 at both ends of Tn7, no transposition is detected. C6 mutations have no effect on transposition frequency. Replacement with 5'-A3C4G5C6G7C8-3' at the right end of Tn7 apparently abolishes transposition; yet in the double mutant, where the inverted repeats are restored by substituting this sequence at both ends of Tn7, transposition is partially rescued. This suggests that the mechanism of Tn7 transposition requires communication between the two ends. Tn7 transposition has always been seen to generate a 5-bp target duplication. This is presumed to result from a staggered cut, plus repair synthesis during transposition. We found that two of our right-end mutants, C2 and C6, sometimes yielded a 6-bp target duplication. This observation implies that cleavage of the target site might also involve interaction with the donor ends which, when mutant, relax the specificity for target-site cleavage.
EL DOCTOR CONRAD LICHTENSTEIN, CIENTIFICO DEL CENTRO DE BIOTECNOLOGIA DEL IMPERIAL COLLEGE DEL LO... more EL DOCTOR CONRAD LICHTENSTEIN, CIENTIFICO DEL CENTRO DE BIOTECNOLOGIA DEL IMPERIAL COLLEGE DEL LONDRES, HA CREADO Y COMENZADO A PROBAR UN NUEVO METODO REVOLUCIONARIO DE PROTECCION DE LOS CULTIVOS CONTRA ENFERMEDADES VIRALES, MEDIANTE EL EMPLEO DE TECNICAS DE MANIPULACION GENETICA.
The Plant Cell Online, 1992
Homologous recombination has been extensively studied in bacteria, yeast, and more recently in an... more Homologous recombination has been extensively studied in bacteria, yeast, and more recently in animal cells, but little is known about this process in plants. We present here an analysis of meiotic and somatic chromosomal recombination between closely linked ...
Proceedings of the …, 1996
Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and anim... more Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and animal systems, has surprisingly not been reported for plants. We have discovered geminivirus-related DNA (GRD) sequences, in the form of distinct sets of multiple direct repeats comprising three related repeat classes, situated in a unique locus in the Nicotiana tabacum (tobacco) nuclear genome. The organization of these sequences is similar or identical in eight different tobacco cultivars we have examined. DNA sequence analysis reveals that each repeat has sequences most resembling those of the New World geminiviral DNA replication origin plus the adjacent AL1 gene, encoding the viral replication protein. We believe these GRD sequences originated quite recently in Nicotiana evolution through integration of geminiviral DNA by some combination of the processes of illegitimate recombination, amplification, deletions, and rearrangements. These events must have occurred in plant tissue that was subsequently able to contribute to meristematic tissue yielding gametes. GRD may have been retained in tobacco by selection or by random fixation in a small evolving population. Although we cannot detect transcription of these sequences, this does not exclude the possibility that they may originally have been expressed.
Trends in Biotechnology, 1992
The use of antisense RNA to manipulate plant metabolism has already beell shown to be successful ... more The use of antisense RNA to manipulate plant metabolism has already beell shown to be successful in altering pigment production in flowers and maniptrlating the ripening and softening processes in fruit. The major losses in crop yields due to viral pathogens hFve led to kvestigations of many strategies for developing *virusresistant plants. By inhibiting viral replication, the use of antisense may permit the engineering of such resistanI plants.
Un metodo de estimacion del numero de moleculas de polinucleotidos de partida secuenciadas a part... more Un metodo de estimacion del numero de moleculas de polinucleotidos de partida secuenciadas a partir de multiples muestras, comprendiendo el metodo: unir un adaptador a las moleculas de polinucleotidos de partida de multiples muestras diferentes, en donde el adaptador para cada muestra comprende: una marca de un unico identificador multiplex (MID) especifico para la muestra, haciendo posible la marca del identificador multiplex la identificacion de la muestra a partir de la cual se deriva un polinucleotido; y una region de bases degeneradas (DBR) que comprende al menos una base de nucleotidos seleccionada de: R, Y, S, W, K, M, B, D, H, V, N, y las versiones modificadas de las mismas, siendo la degeneracion de las DBR de tal modo que es probable que cada polinucleotido individual tenga una DBR diferente; reunir las multiples muestras unidas a adaptadores diferentes para generar una muestra conjunta; amplificar los polinucleotidos unidos al adaptador en la muestra conjunta; secuenciar ...
Des aspects de la presente invention concernent des procedes et des compositions pour la determin... more Des aspects de la presente invention concernent des procedes et des compositions pour la determination du nombre de molecules individuelles de polynucleotide issues de la meme region genomique du meme echantillon d'origine qui ont ete sequencees dans une configuration particuliere ou un procede particulier d'analyse de sequence. Dans ces aspects de l'invention, une region de base degeneree (DBR) est fixee aux molecules initiales de polynucleotide qui sont ulterieurement sequencees (par exemple, apres que certaines etapes du procede sont realisees, par exemple, l'amplification et/ou l'enrichissement). Le nombre de sequences DBR differentes presentes dans un cycle de sequencage peut etre utilise pour determiner/estimer le nombre de polynucleotides initiaux differents qui ont ete sequences. Les DBR peuvent etre utilisees pour augmenter de nombreuses applications differentes d'analyse de sequences d'acides nucleiques, permettant notamment des determinations d...
BMC Proceedings
We are developing novel methods to simultaneously analyze candidate genes or regions from multipl... more We are developing novel methods to simultaneously analyze candidate genes or regions from multiple samples in a single experiment rapidly and cost-effectively. One method, we call Reflex, provides an elegant approach to sample preparation for long-range PCR (LR-PCR) amplicons. Current methods use LR-PCR for target enrichment then use random fragmentation of each sample followed by ligation of DNA barcodes before sequencing: this approach is expensive and labour intensive.