Nobuhiko Takamatsu

Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA.

We have constructed three tobacco mosaic virus (TMV) cDNA derivatives by modification of the full‐length cDNA clone from which infectious TMV‐RNA can be transcribed in vitro. A coatless TMV construct lacks most of the coat protein gene and chimeric TMV constructs retain the bacterial chloramphenicol acetyltransferase (CAT) gene in place of the coat protein gene. When in vitro transcripts from these cDNA derivatives were inoculated on the local lesion tobacco plants, TMV‐specific lesions were produced. In the case of the TMV–CAT chimeras, however, the lesions were small compared to those of wild‐type TMV and those produced by transcript derived from the coatless construct. Northern blot analysis of RNA extracted from the inoculated leaves of the systemic host plants revealed replication of the derivative genomic RNAs and production of their own subgenomic RNAs corresponding to the coat protein mRNA. The TMV–CAT chimeras produced biologically active CAT in the inoculated leaves of the systemic host. CAT activity increased at least until 2 weeks post‐inoculation and was ~0.1 units/mg of tissue at 10 days post‐inoculation. Thus, TMV–RNA may be utilized as a new plant expression vector.

Two concomitant base substitutions in the putative replicase genes of tobacco mosaic virus confer the ability to overcome the effects of a tomato resistance gene, Tm-1

A resistance‐breaking strain of tobacco mosaic virus (TMV), Ltal, is able to multiply in tomatoes with the Tm‐1 gene, unlike its parent strain, L. Comparison of the genomic sequences of L and Lta1 revealed two base substitutions resulting in amino acid changes in the 130 and 180 kd proteins: Gln‐979 → Glu and His‐984 → Tyr. To clarify their involvement in the resistance‐breaking property of Lta1, the two substitions were introduced into L by an in vitro transcription system to generate a mutant strain, T1. T1 multiplied in Tm‐1/Tm‐1 tomatoes with symptoms as did Lta1. Two additional mutant strains were constructed, each of which had one base substitution which caused a His‐984 → Tyr change (T2) or a Gln‐979 → Glu change (T3). T3 multiplied in tomato plants and protoplasts with the Tm‐1 gene, indicating that the single base substitution is sufficient to overcome the resistance. T2 also multiplied, but its multiplication was greatly decreased. Although no sequence changes were detected in any progeny viruses recovered from plants without the Tm‐1 gene, progeny viruses recovered from T2‐ or T3‐ inoculated Tm‐1/Tm‐1 tomatoes contained in most cases viruses with additional second base substitutions. They caused amino acid changes near the mutagenized residues, suggesting that the ability of T3 to overcome the resistance is not the same as that of Lta1. Sequencing of the genomic RNAs of other independently isolated resistance‐breaking strains revealed the same two base substitutions found in the Lta1 RNA. These observations suggest that the two concomitant base substitutions, and possibly also the resulting amino acid changes, guarantee successful replication of these TMV strains in tomatoes containing the Tm‐1 gene. A strong correlation was found between the ability to overcome the resistance and a decrease in local net charge, suggesting the involvement of an electrostatic interaction between the viral 130 and 180 kd proteins and a putative host resistance factor.

Single amino acid substitution in 30K protein of TMV defective in virus transport function

Involvement of the tobacco mosaic virus (TMV) coded 30K protein in a virus transport function within the infected plant has been suggested. Previously a temperature sensitive mutant, TMV Ls 1, that is defective in cell-to-cell movement at a restrictive temperature, was reported. To demonstrate a relationship between the 30K protein and the transport function, the nucleotide sequences of the 30K and coat protein cistrons of the mutant, TMV Ls 1, and the wild type, TMV L (tomato strain) were compared. A single base substitution which causes replacement of a proline codon in the L strain by a serine codon was found in the Ls 1 mutant. Results support the notion that the 30K protein is responsible for the virus transport function.

Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector

To examine the validity of the strategy to express a foreign gene as a fusion protein with the coat protein (CP) of tobacco mosaic virus (TMV), we have constructed ENK RNA by using an in vitro transcription system of TMV RNA. ENK RNA differs from TMV RNA only in that ENK RNA carries an additional sequence coding for Leu‐enkephalin (Tyr‐Gly‐Gly‐Phe‐Leu) (Enk) with a preceding in‐frame methionine just before the termination codon of CP gene. In protoplasts inoculated with ENK RNA, CP+Enk fusion protein accumulated as the major protein.

The 5'-terminal sequence of TMV RNA. Question on the polymorphism found in vulgare strain.

The complete nucleotide sequence of TMV RNA (common strain) reported in [Proc. Natl. Acad. Sci. USA (1982) 79, 5818] its 5′‐end to be represented by two variants which differed in length. We have tested that result and sequenced the 5′‐terminal regions of two strains of TMV RNA (common strain OM and tomato strain L) using cloned cDNA copies. The results showed that the 5′‐terminal region of the TMV genome is not polymorphic and that one of the two variants cited above represents a tomato strain but not the common strain.

Epigenetic regulation of hibernation-associated HP-20 and HP-27 gene transcription in chipmunk liver

The chipmunk hibernation-related proteins (HPs) HP-20 and HP-27 are components of a 140-kDa complex that dramatically decreases in the blood during hibernation. The HP-20 and HP-27 genes are expressed specifically in the liver and are downregulated in hibernating chipmunks. Hibernation-associated physiological changes are assumed to be under genetic control. Therefore, to elucidate the molecular mechanisms of hibernation, here we examined the mechanisms behind the altered HP-20 and HP-27 gene expression in nonhibernating versus hibernating chipmunks. Chromatin immunoprecipitation (ChIP) analyses revealed that histone H3 on the HP-20 and HP-27 gene promoters was highly acetylated at lysine (K) 9 and K14 and highly trimethylated at K4 in the liver of nonhibernating chipmunks, while these active histone modifications were nearly absent in hibernating chipmunks. Furthermore, histone acetyltransferases and a histone methyltransferase were associated with the HP-20 and HP-27 gene promoters primarily in nonhibernating chipmunks. Consistent with a previous finding that HNF-1 and USF can activate HP-20 and HP-27 gene transcription by binding to the proximal promoter region, ChIP-quantitative PCR (qPCR) analyses revealed that significantly less HNF-1 and USF were bound to these gene promoters in hibernating than in nonhibernating chipmunks. These findings collectively indicated that the hibernation-associated HP-20 and HP-27 gene expression is epigenetically regulated at the transcriptional level by the binding of HNF-1 and USF to their proximal promoters, and that histone modification has a key role in hibernation-associated transcriptional regulation.

JLP-JNK signaling protects cancer cells from reactive oxygen species-induced cell death

Oxidative stress, which can be caused by an overproduction of reactive oxygen species (ROS), often leads to cell death. In recent years, c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP, also known as SPAG9 or JIP4), a scaffold protein for JNK mitogen-activated protein kinase (MAPK) signaling pathways, was found to serve as a novel biomarker for cancer. However, although JNK MAPK pathways are reported to be activated in response to various stimuli, including oxidative stress, whether JLP is involved in ROS signaling remains unknown. In this study, we examined the role of JLP in hydrogen peroxide (H2O2)-induced cancer cell death, and found that JLP knockdown (KD) cells exhibit a substantially enhanced cell death response, along with increased intracellular ROS levels. This is the first demonstration of a protective role for JLP in response to cell-death stimulation. We also found that the H2O2-induced JNK activation was attenuated in JLP KD cancer cells. The decreases in cell viability and JNK activation in the JLP KD cells were almost completely reversed by expressing wild-type JLP, but not a mutant JLP lacking the JNK-binding domain. These data collectively suggest that the JLP-JNK signaling pathway counteracts ROS-induced cancer cell death.