Masato Ikegami

The Nucleotide Sequence and Genome Structure of Mung Bean Yellow Mosaic Geminivirus

Complete nucleotide sequences of the infectious cloned DNA components (DNA 1 and DNA 2) of mung bean yellow mosaic virus (MYMV) were determined. MYMV DNA 1 and DNA 2 consists of 2,723 and 2,675 nucleotides respectively. DNA 1 and DNA 2 have little sequence similarity except for a region of approximately 200 bases which is almost identical in the two molecules. Analysis of open reading frames revealed nine potential coding regions for proteins of mol. wt. > 10,000, six in DNA 1 and three in DNA 2. The nucleotide sequence of MYMV DNA was compared with that of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and African cassava mosaic virus (ACMV). The 200‐base region common to the two DNAs of each virus had little sequence similarity, except for a highly conserved 33‐36 base sequence potentially capable of forming a stable hairpin structure. The potential coding regions in the MYMV DNAs had counterparts in the BGMV, TGMV and ACMV, suggesting an overall similarity in genome organization, except for absence of 1L3 in MYMV DNA 1. The most highly conserved ORFs, MYMV 1R1, BGMV 1R1, TGMV 1R1 and ACMV 1R1, are the putative genes for the coat proteins of MYMV, BGMV, TGMV and ACMV, respectively. MYMV 1L1 has also a high degree of sequence similarity with BGMV 1L1, TGMV 1L1 and ACMV 1L1.

Complete Nucleotide Sequence and the Genome Organization of Patchouli Mild Mosaic Virus RNA1

The nucleotide sequence of the RNA1 of Patchouli mild mosaic virus (PatMMV) has been determined. It contains 5,957 nucleotides excluding the 3′-terminal poly(A) tail and contains a single long open reading frame (ORF) of 5,613 nucleotides extending from nucleotide 235 to 5847. The predicted polyprotein encoded by the long ORF is 1,870 amino acids in length with a molecular weight of 210 kD. The conserved residues of RNA-dependent RNA polymerase, cysteine protease, purine NTP-binding domain and a cofactor for viral protease are present in a 210-kD polyprotein. As PatMMV RNA showed high sequence identity (81–97%) with BBWV-2 RNA, PaMMV may be one strain of BBWV-2.

E. coli spheroplast-mediated transfer of cloned cauliflower mosaic virus DNA into plant protoplasts

SummarySaishin (Brassica chinensis L.) mesophyll protoplasts and E. coli spheroplasts harbouring hybrid plasmid with tandemly dimerized cauliflower mosaic virus DNA were mixed in ratios of 1:1,000 and incubated for 20 min at 30° C in the presence of 20% polyvinyl alcohol. Subsequently, protoplasts/spheroplasts mixture was washed with high pH-high Ca buffer. After 3 days of culture, 8% of Saishin protoplasts were transfected as monitored by immunofluorescence technique. When plant protoplasts and bacterial spheroplasts were mixed in ratios of 1:100 or 1:2,000, 1% or 5% of protoplasts were transfected, respectively.

Complete Nucleotide Sequence and the Genome Organization of Tobacco Leaf Curl Geminivirus from Japan

The genomic DNA of tobacco leaf curl geminivirus (TLCV) from tomato plants with leaf curl disease in Japan has been sequenced. The single circular DNA molecule comprises 2,761 nucleotides. TLCV DNA contains six open reading frames (ORFs) capable of encoding proteins with a molecular weight greater than 10 K. In total nucleotide sequence comparisons with other geminiviruses, TLCV was most closely related to tomato leaf curl virus from Taiwan (TwToLCV) (76% identity), tomato leaf curl virus from Bangalore (ToLCV‐Ba) (74%) and agerantum yellow vein virus (AYVV) (74%), all possessing a monopartite genome. The significant but relatively low sequence similarity in the genomic DNA between TLCV and other geminiviruses suggests it is a distinct geminivirus in genus Begomovirus.

Physical mapping and molecular cloning of mung bean yellow mosaic virus DNA

Viral single-stranded DNA of mung bean yellow mosaic virus (MYMV) was converted to the double-stranded state in vitro, and physical mapping was carried out. The genome of MYMV was found to consist of two major components (designated as DNA 1 and DNA 2). In addition, some minor components were detected. Molecular cloning of the major components was carried out, using in vitro double-stranded DNA and replicative intermediate DNAs. DNA 1 is about 2.72 and DNA 2 about 2.67 kilobase pairs. No similarities were observed when the two restriction maps of DNA 1 and 2 were compared.

Total Nucleotide Sequences of the Infectious Cloned DNAs of Bean Golden Mosaic Virus

Complete nucleotide sequences of the infectious cloned DNA components (DNA A and B) of bean bolden mosaic virus were determined. The DNA A (2585 nucleotides) and DNA B (2647 nucleotides) have little sequence homology with each other, but both A and B contain a common region of 205 nucleotides. A possible large hairpin structure is detected in the common region. Nucleotide sequences of DNAs A and B revealed the presence of 8 potential coding regions for proteins (m.w.> 10,000). Among them, four open reading frames (ORFs 1–4) encode proteins of m.w. 30,000 or greater, and are individually coded in virion DNA A and B senses (+) and their complementary senses (‐), respectively. The other four ORFs 5–8 are in virion DNA B(+) and its complementary sense (‐). All of the ORFs 1–4 have regulatory signals for RNA synthesis (TATAA/T) in the region 5′ upstream from a potential start codon ATG.

Potential gene products of bean golden mosaic virus have higher sequence homologies to those of tomato golden mosaic virus than to those of cassava latent virus.

Comparison of the nucleotide sequences of the DNAs of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and cassava latent virus (CLV) revealed a fairly close relationship between BGMV DNA1, TGMV DNA1, and CLV DNA1 and a comparatively distant relationship between BGMV DNA2, TGMV DNA2, and CLV DNA2. The 200-base region common to the two DNAs of each virus had little sequence homology, except for a highly conserved 33–36 base sequence potentially capable of forming a stable hairpin structure. All the potential coding regions in the BGMV DNAs had counterparts in the TGMV and CLV DNAs suggesting an overall similarity in genome organization but two potential coding regions in the BGMV DNAs had no counterparts in the TGMV DNAs. The most highly conserved ORFs, BGMV 1R1, TGMV 1R1, and CLV 1R1, are the putative genes for the coat proteins of BGMV, TGMV, and CLV. BGMV 1R1 has 91.9% and 71.6% homology with respect to TGMV and CLV. The ORFs (BGMV 1L1; CLV 1L1; TGMV 1L1) and the two smaller overlapping ORFs (BGMV 1L2, 1L3; TGMV 1L2, 1L3; CLV 1L5, 1L3) are conserved in the three viruses. BGMV 2R1 and BGMV 2L1 have higher homology with respect to TGMV but not with respect to 2R1 and 2L1 in CLV. From this study we conclude that BGMV is more closely related to TGMV than CLV.

Identification of the Coat Protein Gene of Bean Golden Mosaic Geminivirus

An open-reading frame designated 1R1, on DNA component 1 of bean golden mosaic geminivirus (BGMV), has been identified as the coat protein gene. A DraI restriction fragment of BGMV DNA1 that includes 1R1 was inserted into the SmaI pKK223-3 expression vector. The 32 kD protein expressed in Escherichia coli cells reacted with antibodies to the BGMV capsid polypeptide and behaved identically to purified capsid protein in western blots.

Infectivity of virus-specific double-stranded DNA from tissue infected by mung bean yellow mosaic virus

Abstract A double-stranded (ds) DNA which may be a replicative intermediate was isolated from bean ( Phaseolus vulgaris L. ‘Top Crop’) leaves systemically infected with mung bean yellow mosaic virus. The isolation method used phenol/chloroform extraction, hydroxyapatite column chromatography, and rate-zonal centrifugation. The dsDNA was a circular molecule and had sequences complementary to those of viral DNA. These molecules infected bean plants and typical symptoms of downward curling appeared.

The complete nucleotide sequence of odontoglossum ringspot virus (Cy-1 strain) genomic RNA.

The complete nucleotide sequence of the genomic RNA of odontoglossum ringspot virus Cy‐1 strain (ORSV Cy‐1) was determined using cloned cDNA. This sequence is 6611 nucleotides long containing four open reading frames, which correspond to 126 K, 183 K, 31 K, and 18 K proteins. Its genomic organization is similar to other tobamoviruses, TMV‐V(vulgare), TMV‐L (tomato strain), tobacco mild green mosaic virus (TMGMV) and cucumber green mottle mosaic virus (CGMMV). The 5′ non‐coding regions of ORSV Cy‐1 is 62 nucleotides. The ORFs encoded a 126 K polypeptide and a 183 K read‐through product in which helicase‐sequence and polymerase‐sequence motifs are found. The ORFs encoding the 126 K and 183 K proteins have 61% and 63% identities with those of TMV‐V. The third ORF encoded a 31 K protein homologous to TMV cell‐to‐cell movement protein. It has 63% identities with that of TMV‐V. The fourth ORF encoded an 18 K coat protein. The 5′ non‐coding region, which extends from base 1 to 62 has 2 G residues and a ribosome binding site (AUU). The 3′ non‐coding region, 414 nucleotides in length, is entirely different from that of other tobamoviruses.