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In May 1996, an outbreak of sugarcane mosaic virus (SCMV) was detected in Florida affecting a major commercial sugarcane (Saccharum spp. hybrid) cultivar, CP 72-2086. Identification of SCMV was confirmed by enzyme-linked immunosorbent assay (ELISA) with SCMV antiserum (ATCC no. PVAS 115), and by reverse transcription(RT)-PCR (1,2). The strain of SCMV was identified as E with an RT-PCR-based restriction fragment length polymorphism analysis (1). Although SCMV strain E has been detected in Florida for many decades, it had been confined primarily to S. officinarum clones, to occasional susceptible sugarcane clones in breeding programs, and to various grasses throughout the sugarcane production area. CP 72-2086 plus seven other sugarcane cultivars and 10 sorghum cultivars reacted similarly to inoculation with the isolate of SCMV found recently on CP 72-2086 and an isolate of SCMV collected in 1986. CP 72-2086 was released in 1982 and currently is the second most widely grown cultivar, constituting 18.0% of the commercial sugarcane area in Florida. At the epicenter of infection, located 11 km southeast of Canal Point, over 50% of CP 72-2086 plants had SCMV symptoms. The incidence of mosaic decreased rapidly away from the epicenter. No SCMV was observed in the western area of the sugarcane production area, west of South Bay, FL. References: (1) T. E. Mirkov and J. E. Irvine. Sugar y Azucar 9:23, 1996. (2) G. R. Smith et al. Plant Dis. 78:557, 1994.

The specificity and sensitivity of polymerase chain reaction (PCR) primers developed for ‘Candidatus Liberibacter solanacearum’ and ‘Candidatus Liberibacter psyllaurous’ were evaluated in conventional and real-time PCR assays. All PCR primers were specific for ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ insomuch as they did not detect other prokaryotic plant pathogens that affect potato except for the putative pathogens associated with psyllid-yellows and haywire. Conventional PCR assays were capable of detecting 0.19 to 1.56 ng of total DNA per reaction, and real-time PCR was found capable of detecting 1.56 to 6.25 ng of total DNA per reaction, depending on the specific PCR primer set used. ‘Ca. Liberibacter’ species associated with zebra complex disease (ZC) was confirmed in plants affected by this disease throughout Texas from 2005 to 2008, in seed tubers produced in Wyoming in 2007, and in Colorado, Kansas, Nebraska, and Mexico in 2008. A multiplex PCR assay using ‘Ca. L. solanacearum’–specific primers and primers specific for the β-tubulin DNA regions from potato was developed, providing possible utility of the multiplex assay for ‘Ca. Liberibacter’ detection in different solanaceous plant species. Preliminary studies suggest silverleaf nightshade (Solanum elaeagnifolium), wolfberry (Lycium barbarum), black nightshade (S. ptychanthum), and jalapeno pepper (Capsicum annuum) as additional solanaceous hosts for the ZC-associated bacterium. The ‘Ca. Liberibacter’ species detected in all samples divided into two clusters sharing similarity of 99.8% in their partial 16S rRNA gene sequences and 99.3% in their partial intergenic spacer region (ISR)-23S rRNA gene sequences. Genetic variation in the 16S rDNA region consistently matched that of the ISR-23S rDNA region. In this partial 16S-ISR-23S rDNA region, there was a total of eight single nucleotide polymorphisms among ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ “strains” investigated in this study. ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’ were shown to be very closely related bacteria, if not the same, by successful amplification using a combination of forward primer of ‘Ca. L. solanacearum’ and reverse primer of ‘Ca. L. psyllaurous’ in ZC-affected potato samples. This finding clarifies the current taxonomic status of ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’. The detection of ‘Ca. L. solanacearum’ from haywire-symptomatic potato samples demonstrates that this bacterium might also be associated with this disease.

Increased industrial use of sugarcane (Saccharum spp. hybrid) for food and bioenergy has led to considerable improvements in its genetic transformation, which allowed the development of not only pest- and herbicide-resistant lines but also lines expressing high-value bioproducts and biopolymers. However, the economic benefits of using inexpensive transgenic plant systems for the production of industrial proteins could be offset by high downstream processing costs. In this work, transgenic sugarcane expressing recombinant bovine lysozyme (BvLz) was used to evaluate the feasibility of extraction and fractionation of recombinant proteins expressed in sugarcane stalks. Three pH levels (4.5, 6.0 and 7.5) and three salt concentrations (0, 50, and 150 mM NaCl) were tested to determine BvLz and total protein extractability. Two extraction conditions were selected to prepare BvLz extracts for further processing by cross-flow filtration, a suitable method for concentration and conditioning of extracts for direct applications or prior to chromatography. Partial removal of native proteins was achieved using a 100 kDa membrane but 20-30 % of the extracted BvLz was lost. Concentration of clarified extracts using a 3 kDa membrane resulted in twofold purification and 65 % recovery of BvLz. Loading of concentrated sugarcane extract on hydrophobic interaction chromatography (HIC) resulted in 50 % BvLz purity and 69 % recovery of BvLz.

A survey of cultivated hybrid sugarcane (Saccharum inter-specific hybrid) and noble sugarcane (Saccharum officinarum) in southern China for the presence of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV) and Sugarcane streak mosaic virus (SCSMV) was conducted by RT-PCR from the years 2003 to 2006. SCMV and SrMV, but not SCSMV, were found. A high incidence of SCMV and SrMV coinfection was revealed in both hybrid and noble sugarcanes. All coinfected plants showed mosaic symptom, whereas plants infected with a single virus were symptomatic or asymptomatic. It appears that virus mixtures are more virulent than single infections. The nucleotide sequences of the coat protein (CP) gene of 33 SCMV and 10 SrMV isolates from this study were compared to those of CP genes of SCMV and SrMV reported in GenBank. One hundred and seventy-three SCMV isolates, with the exception of MDB and Abaca strains, can be grouped into five groups, which include three previously known groups, the sugarcane (SCE), maize (MZ), and Thailand groups, and two newly identified groups, the noble sugarcane (NSCE) and Brazil groups. Twenty-two SrMV isolates were divided into two groups, HS (hybrid sugarcane) and NS (noble sugarcane) groups. Five out of eight SrMV hybrid isolates belonged to the HS group, and two SrMV noble isolates and three hybrid isolates were within the NS group. Interestingly, the three hybrid isolates within the NS group were isolated from hybrid sugarcane co-infected with SCMV. This indicates that SCMV helps the NS group SrMV to infect hybrid sugarcane.

In spite of the importance of Citrus in agriculture and recent progress in genetic mapping and cytogenetics of this group, chromosome mapping of Citrus species is still limited to rDNA probes. In order to obtain a better chromosome characterization of one species from this group, CMA/DAPI double staining followed by in situ hybridization using 45S rDNA and 24 BACs (BAC-FISH) were used on Poncirus trifoliata. The BACs used were obtained from a genomic library of this species and were selected by membrane hybridization using genomic DNA. Four of them were isolated from the Citrus tristeza virus (Ctv) resistance gene region. The P. trifoliata karyotype is composed of two chromosome pairs with one terminal and one proximal CMA+ band (B type chromosomes), four chromosome pairs with a single CMA+ band (D type) and three chromosome pairs without bands (F type). In situ hybridization with 13 of the BACs gave single copy signals on seven chromosome pairs. At least one BAC was mapped on each arm of the two B chromosome pairs. Among the four D chromosome pairs, two were identified by BACs mapped on the long arms, one has a 45S rDNA site and the other had no signal. Six BACs allowed identification of the three F chromosome pairs, with one pair hybridizing with four BACs from the Ctv resistance gene region. In summary, all nine chromosome pairs could be differentiated, seven of them by BAC-FISH, while the other two chromosomes could be recognized by the CMA+ band pattern and 45S rDNA sites. This first BAC-FISH map gives a general framework for comparative genome structure and evolutionary studies in Citrus and Poncirus, allowing the integration of genetic and physical maps when these BACs are included.

The genetic diversity of sugarcane yellow leaf virus (SCYLV) was analyzed with 43 virus isolates from Réunion Island and 17 isolates from world-wide locations. We attempted to amplify by reverse-transcription polymerase chain reaction (RT-PCR), clone, and sequence four different fragments covering 72% of the genome of these virus isolates. The number of amplified isolates and useful sequence information varied according to each fragment, whereas an amplicon was obtained with diagnostic primers for 59 out of 60 isolates (98%). Phylogenetic analyses of the sequences determined here and additional sequences of 11 other SCYLV isolates available from GenBank showed that SCYLV isolates were distributed in different phylogenetic groups or belonged to single genotypes. The majority of isolates from Réunion Island were grouped in phylogenetic clusters that did not contain any isolates from other origins. The complete six ORFs (5612 bp) of five SCYLV isolates (two from Réunion Island, one from Brazil, one from China, and one from Peru) were amplified, cloned, and sequenced. The existence of at least three distinct genotypes of SCYLV was shown by phylogenetic analysis of the sequences of these isolates and additional published sequences of three SCYLV isolates (GenBank accessions). The biological significance of these genotypes and of the origin of the distinct lineage of SCYLV in Réunion Island remains to be determined.

The effects of the snowdrop lectin, Galanthus nivalis agglutinin (GNA), delivered through an artificial diet, on growth, development, and life history parameters of the Mexican rice borer, Eoreuma loftini (Dyar), were evaluated in the laboratory. Incorporation of GNA at three treatment levels, 0.5, 1.0, and 2.0% of total dietary protein, in the larval diet significantly decreased larval survivorship and percentage of adults emerging relative to a control diet lacking GNA, whereas differences were not observed among the three treatment levels. Both larvae and pupae in the control were 8-25% larger than those in the GNA treatments, but differences were not observed between larvae in the GNA treatments. Furthermore, presence of GNA did not affect larval and pupal developmental periods, longevities, and fecundities compared with the control. Mexican rice borer life history parameters, such as net reproductive rate and intrinsic rate of increase, were substantially reduced by the presence of GNA in the diet, but differences were not evident among the three GNA treatment levels.

Location and parasitism of Diatraea saccharalis (F.) by Cotesia flavipes (Cameron) were compared between hosts fed either conventional or transgenic sugarcane expressing Galanthus nivalis L. agglutinin (GNA) under choice and no-choice conditions. In olfactometer experiments, females of C. flavipes randomly visited the different odor zones available but spent significantly more time in odor zones corresponding to plants damaged by D. saccharalis versus blank (control) odor zones. However, they spent similar amounts of time in odor zones corresponding to damaged transgenic and conventional sugarcane. Laboratory and field-cage experiments showed that C. flavipes equally parasitized D. saccharalis feeding on transgenic and conventional sugarcane plants. Moreover, sex ratio and brood size of C. flavipes were similar on hosts fed transgenic or conventional sugarcane. The results of this study suggest that transgenic sugarcane expressing GNA would not significantly affect host location and parasitism of D. saccharalis by C. flavipes in the field.

The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45 696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.Key words: Poncirus, citrus tristeza virus, chromosome walk, resistance gene.

Isolation of the terminal portions of genomic DNA cloned in bacterial artificial chromosomes (BACs) is an important step in map-based cloning, and several methods have been developed. Here, we present a new method based on double-restriction-enzyme digestion followed by anchored PCR. BAC DNA was digested with two enzymes: NotI and one of four enzymes (EcoRV, HpaI, StuI, or XmnI) that produce blunt termini. After dephosphorylation, these digestions were ligated to NotI- and EcoRV-digested pMSK, a new cloning vector developed in this work that is derived from pBluescript SK(+). PCR products representing the left- and right-terminal sequences of BAC inserts were obtained using a primer complementary to pMSK and a primer complementary to sequences in either the left arm or the right arm of the BAC vector pBeloBAC11. We have tested this method with 15 different BAC clones, and PCR products representing both the left- and right-terminal sequences have been obtained from all 15 BAC clones. This method is simple, fast, reproducible, and uses the same set of primers for any restriction enzyme used. With some modifications, it can also be used for isolating the terminal portions of genomic DNA cloned in yeast artificial chromosomes and P1-derived artificial chromosomes.Key words: BAC, anchored PCR, terminal sequence isolation, chromosome walk.

We have derived the genomic nucleotide sequence of an emerging virus, the Sugarcane yellow leaf virus (ScYLV), and shown that it produces one to two subgenomic RNAs. The family Luteoviridae currently includes the Luteovirus, Polerovirus, and Enamovirus genera. With the new ScYLV nucleotide sequence and existing Luteoviridae sequence information, we have utilized new phylogenetic and evolutionary methodologies to identify homologous regions of Luteoviridae genomes, which have statistically significant altered nucleotide substitution ratios and have produced a reconstructed phylogeny of the Luteoviridae. The data indicate that Pea enation mosaic virus-1 (PEMV-1), Soybean dwarf virus (SbDV), and ScYLV exhibit spatial phylogenetic variation (SPV) consistent with recombination events that have occurred between poleroviral and luteoviral ancestors, after the divergence of these two progenitor groups. The reconstructed phylogeny confirms a contention that a continuum in the derived sequence evolution of the Luteoviridae has been established by intrafamilial as well as extrafamilial RNA recombination and expands the database of recombinant Luteoviridae genomes that are currently needed to resolve better defined means for generic discrimination in the Luteoviridae (D'Arcy, C. J. and Mayo, M. 1997. Arch. Virol. 142, 1285-1287). The analyses of the nucleotide substitution ratios from a nucleotide alignment of Luteoviridae genomes substantiates the hypothesis that hot spots for RNA recombination in this virus family are associated with the known sites for the transcription of subgenomic RNAs (Miller et al. 1995. Crit. Rev. Plant Sci. 14, 179-211), and provides new information that might be utilized to better design more effective means to generate transgene-mediated host resistance.

Sugarcane mosaic is the most widespread virus disease affecting sugarcane production. We have established a collection of seven prominent sugarcane mosaic potyvirus (SCMV) strains currently causing disease in sugarcane throughout the world and originally found in sugarcane in the United States. This collection includes SCMV strains A, B, D, and E, and the sorghum mosaic virus (SrMV) strains SCH, SCI, and SCM. These viruses were propagated on Sorghum bicolor cv. Rio and purified. Cloned cDNAs representing 2.0 kb of the 3′ termini, obtained after a reversetranscriptase-polymerase chain reaction (RT-PCR) on purified virions using an oligo(dT) primer and degenerate primers with sequences located in the NIb gene, have been sequenced for each of these strains. A comparative analysis of the deduced amino acid sequences in the NIb and coat protein genes and of the nucleotide sequences in the 3′-untranslated region, among these seven viruses and among six other members of the SCMV subgroup, confirms that there are at least four, but suggests that there are five, distinct viruses in the SCMV complex. Based on these seven new sequences and on the available sequence data for six other members of the SCMV subgroup, we have developed group-specific primers for use in RT-PCR-based restriction fragment length polymorphism analysis for rapid discrimination between strains of SCMV and SrMV. This is the first assay for differentiating strains of SCMV and SrMV that does not require interpretation of symptoms on differential hosts.

In spite of the importance of Citrus in agriculture and recent progress in genetic mapping and cytogenetics of this group, chromosome mapping of Citrus species is still limited to rDNA probes. In order to obtain a better chromosome characterization of one species from this group, CMA/DAPI double staining followed by in situ hybridization using 45S rDNA and 24 BACs (BAC-FISH) were used on Poncirus trifoliata. The BACs used were obtained from a genomic library of this species and were selected by membrane hybridization using genomic DNA. Four of them were isolated from the Citrus tristeza virus (Ctv) resistance gene region. The P. trifoliata karyotype is composed of two chromosome pairs with one terminal and one proximal CMA(+) band (B type chromosomes), four chromosome pairs with a single CMA(+) band (D type) and three chromosome pairs without bands (F type). In situ hybridization with 13 of the BACs gave single copy signals on seven chromosome pairs. At least one BAC was mapped on each arm of the two B chromosome pairs. Among the four D chromosome pairs, two were identified by BACs mapped on the long arms, one has a 45S rDNA site and the other had no signal. Six BACs allowed identification of the three F chromosome pairs, with one pair hybridizing with four BACs from the Ctv resistance gene region. In summary, all nine chromosome pairs could be differentiated, seven of them by BAC-FISH, while the other two chromosomes could be recognized by the CMA(+) band pattern and 45S rDNA sites. This first BAC-FISH map gives a general framework for comparative genome structure and evolutionary studies in Citrus and Poncirus, allowing the integration of genetic and physical maps when these BACs are included.

The effects of the snowdrop lectin, Galanthus nivalis agglutinin (GNA), delivered through an artificial diet, on growth, development, and life history parameters of the Mexican rice borer, Eoreuma loftini (Dyar), were evaluated in the laboratory. Incorporation of GNA at three treatment levels, 0.5, 1.0, and 2.0% of total dietary protein, in the larval diet significantly decreased larval survivorship and percentage of adults emerging relative to a control diet lacking GNA, whereas differences were not observed among the three treatment levels. Both larvae and pupae in the control were 8-25% larger than those in the GNA treatments, but differences were not observed between larvae in the GNA treatments. Furthermore, presence of GNA did not affect larval and pupal developmental periods, longevities, and fecundities compared with the control. Mexican rice borer life history parameters, such as net reproductive rate and intrinsic rate of increase, were substantially reduced by the presence of GNA in the diet, but differences were not evident among the three GNA treatment levels.

The impact of snowdrop lectin (Galanthus nivalis agglutinin, GNA) expressed in transgenic sugarcane on life history parameters of Mexican rice borer [Eoreuma loftini (Dyar)] and sugarcane borer [Diatraea saccharalis (F.)] (both Lepidoptera: Pyralidae) was evaluated. In the laboratory, lyophilized sugarcane leaf sheath tissue was incorporated in a meridic diet resulting in a GNA concentration of 0.47% of total protein, and used for insect bioassays over two successive generations. Deleterious effects of GNA were not observed on survival, weight, and developmental periods of larvae and pupae, nor on adult fecundity and egg viability of D. saccharalis. Moreover, in the first generation, addition of transgenic sugarcane tissue to the diet enhanced larval growth in D. saccharalis resulting in higher larval and pupal weight compared with diet with nontransgenic sugarcane, but this effect was not observed in the second generation. In contrast, larval survival, percent adult emergence, and female fecundity of E. loftini were significantly reduced when fed transgenic sugarcane diet compared with nontransgenic sugarcane diet. In addition, a substantial reduction of female pupal weight of E. loftini was observed in the second generation. For both species, the only consistent effect of GNA in both generations was a reduction in adult female longevity. Life table parameters showed that GNA at the level found in the transgenic diet negatively affected development and reproduction of E. loftini, whereas it had a nil to positive effect on development and reproduction of D. saccharalis.

The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45,696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.

Transgenic sugarcane parents containing multiple copies of herbicide resistance ( bar) and Sorghum mosaic virus (SrMV) resistance ( hut) genes were crossed with non-transgenic sugarcane varieties. Segregation of the transgenes in the progeny was determined using Southern blot analysis; herbicide resistance and SrMV resistance were assessed using bioassays. The segregation data were used to infer linkage relationships between transgenes in the parent plants. In two of the parents, all transgene insertions were linked in one position in the genome, although some recombination between insertion events did occur. In the third parent, insertion had occurred in two independent, unlinked loci. Analysis of progeny of this parent indicated that rearrangement or mutation occurred in both loci, resulting in non-parental transgene DNA fragments in some progeny. Most transgenic progeny containing the bar gene showed resistance to herbicide. SrMV inoculation indicated that a fairly high proportion of the transgenic progeny showed susceptibility. As the post-transcriptional gene silencing mechanism responsible for the virus resistance phenotype may be reset during meiosis, phenotypic screening of older plants may be a more reliable indication of virus resistance than screening young seedlings. This is the first report of transgene segregation in sugarcane, and we have demonstrated that transgenic sugarcane parents showing stable inheritance of transgenes can be effectively used in breeding programs.

Isolation of the terminal portions of genomic DNA cloned in bacterial artificial chromosomes (BACs) is an important step in map-based cloning, and several methods have been developed. Here, we present a new method based on double-restriction-enzyme digestion followed by anchored PCR. BAC DNA was digested with two enzymes: NotI and one of four enzymes (EcoRV, HpaI, StuI, or XmnI) that produce blunt termini. After dephosphorylation, these digestions were ligated to NotI- and EcoRV-digested pMSK, a new cloning vector developed in this work that is derived from pBluescript SK(+). PCR products representing the left- and right-terminal sequences of BAC inserts were obtained using a primer complementary to pMSK and a primer complementary to sequences in either the left arm or the right arm of the BAC vector pBeloBAC11. We have tested this method with 15 different BAC clones, and PCR products representing both the left- and right-terminal sequences have been obtained from all 15 BAC clones. This method is simple, fast, reproducible, and uses the same set of primers for any restriction enzyme used. With some modifications, it can also be used for isolating the terminal portions of genomic DNA cloned in yeast artificial chromosomes and P1-derived artificial chromosomes.

The complete sequence (19,249 nucleotides) of the genome of citrus tristeza virus (CTV) isolate SY568 was determined. The genome organization is identical to that of the previously determined CTV-T36 and CTV-VT isolates. Sequence comparisons revealed that CTV-SY568, a severe stem-pitting isolate from California, has more than 87% overall sequence identity with CTV-VT, a seedling yellows isolate from Israel. Although SY568 has an overall sequence identity of 81% with CTV-T36, a quick decline isolate from Florida, the sequence identity in the 3' half of the genome is over 90% while the sequence identity in the 5' half of the genome is as low as 56%. Based on the sequence alignments of these three isolates, sequences in the 3' half of the genome are generally well conserved, while the sequences in the 5' half are relatively divergent. Sequence data of independent overlapping clones from the CTV-SY568 genome revealed two regions with highly divergent sequences. In open reading frame 1b (RNA dependent RNA polymerase), there were 118 nucleotide differences that lead to 16 amino acid changes. In the open reading frame of the divergent coat protein gene, 5 amino acid changes result from 48 nucleotide differences. Most differences occurred in the third position of the codons, and resulted in silent amino acid substitutions. RNase protection assays demonstrated that most of the clones obtained are representative of the major RNA species of this isolate. Northern analysis indicated that CTV-SY568 accumulated more viral RNA including genomic and certain subgenomic RNAs than isolates VT or T36 in sweet orange.

A simple and fast RNA gel blot procedure is described that uses 50 mM NaOH to simultaneously transfer and fix RNA to a positively charged nylon membrane. The RNA is transferred in a downward direction, and transfer is routinely completed within 2.5 h. The resulting blots give increased sensitivity over existing methods without affecting RNA integrity and can be used in both radioactive and nonradioactive detection procedures.