Mammalian spermatogenesis occurs in a precise and coordinated manner in the seminiferous tubules. One of the attempts to understand the detailed biological process during mammalian spermatogenesis at the molecular level has been to identify the testis specific genes followed by study of the testicular expression pattern of the genes. From the subtracted cDNA library of rat testis prepared using representational difference analysis (RDA) method, a complimentary DNA clone encoding type III member of a DnaJ family protein, DnaJC18, was cloned (GenBank Accession No. DQ158861). The fulllength DnaJC18 cDNA has the longest open reading frame of 357 amino acids. Tissue and developmental Northern blot analysis revealed that the DnaJC18 gene was expressed specifically in testis and began to express from postnatal week 4 testis, respectively. In situ hybridization studies showed that DnaJC18 mRNA was expressed only during the maturation stages of late pachytene, round and elongated spermatids of adult rat testis. Western blot analysis with DnaJC18 antibody revealed that 41.2 kDa DnaJC18 protein was detected only in adult testis. Immunohistochemistry study further confirmed that DnaJC18 protein, was expressed in developing germ cells and the result was in concert with the in situ hybridization result. Confocal microscopy with GFP tagged DnaJC18 protein revealed that it was localized in the cytoplasm of cells. Taken together, these results suggested that testis specific DnaJC18, a member of the type III DnaJ protein family, might play a role during germ cell maturation in adult rat testis.

Arabidopsis atDjC53 and atDjC32 gene DnaJ-like protein homologous to DnaJ-like protein was characterized for the functional analysis of DnaJ-like protein. It was shown that atDjC53 and atDjC32 RNA expression is induced by heat shock stress and atDjC53- and atDjC32-GFP was targeted to the nucleus of protoplasts. The atDjC53 and atDjC32 promoter (1 kb) was isolated and fused to the GUS reporter gene to investigate gene regulation of atDjC53 and atDjC32 specific to heat shock stress or to developmental organ in the transgenic lines. RNAi and overexpression construct was employed to generate atDjC53 and atDjC32 knock-out plants for the study of their function. Molecular function of atDjC53 and atDjC32 is discussed in relation to heat shock and also developmental stages in Arabidopsis.

Arabidopsis atDjC53 and atDjC32 gene DnaJ-like protein homologous to DnaJ-like protein was characterized for the functional analysis of DnaJ-like protein. It was shown that atDjC53 and atDjC32 RNA expression is induced by heat shock stress and atDjC53- and atDjC32-GFP was targeted to the nucleus of protoplasts. The atDjC53 and atDjC32 promoter (1 kb) was isolated and fused to the GUS reporter gene to investigate gene regulation of atDjC53 and atDjC32 specific to heat shock stress or to developmental organ in the transgenic lines. RNAi and overexpression construct was employed to generate atDjC53 and atDjC32 knock-out plants for the study of their function. Molecular function of atDjC53 and atDjC32 is discussed in relation to heat shock and also developmental stages in Arabidopsis.

We have isolated wound-inducible genes from soybean using suppression subtractive hybridization (SSH) method and were able to obtain the full-length clone of GmDjp1 gene encoding DnaJ-like protein. The full-length cDNA of GmDjp1 is 689 bp with an open reading frame (ORF) consisting of 163 amino acid (aa). Genomic southern blot confirmed that soybean genome has two copies of GmDjp1 gene. Northern blot analysis showed that the RNA expression of GmDjp1 gene is specifically induced by heat, NaCl, wounding and drought stresses. It was demonstrated that GmDjp1-GFP was targeted to the nucleus in tobacco cell. GmDjp1 overexpression plants showed more susceptible to salt and heat stress compared to WT. RNA expression level of Hsp18.2 and Hsp25.3-P was lower than that of WT during recovery after heat hock in plants. This indicates that GmDjp1 may play a negative regulator to stress responses in plants.

We isolated wound-inducible genes using suppression subtractive hybridization (SSH) method and were able to obtain to clone W3 gene encoding dnaJ like protein. The full-length cDNA of W3 is 689 bp with an open reading frame (ORF) consisting of 163 amino acid (aa). Genomic southern blot confirmed that soybean genome has two copies of W3 gene. Northern blot analysis was also carried out for the gene expression during heat, NaCl, drought, wounding stresses. The expression of W3 gene specifically induced by heat, NaCl, wounding and drought stress. Using GFP fusion vector, W13-GFP was targeted both to nucleus. For the functional analysis of W3, His-tagged W3 recombinant protein was heterologously expressed in E. coli. The W3 recombinant cells showed enhanced heat tolerance compared to that of vector control cells. We suggest that dnaJ-like W3 protein function as molecular chaperone in the nucleus of the plant cell during various stresses.

We isolated wound-inducible genes using suppression subtractive hybridization (SSH) method and were able to obtain to clone w123 gene encoding dnaJ like protein. The full-length cDNA of w123 is 689 bp with an open reading frame (ORF) consisting of 163 amino acid (aa). Genomic southern blot confirmed that soybean genome has two copies of w123 gene. Northern blot analysis was also carried out for the gene expression during heat, NaCl, drought, wounding stresses. The expression of w123 gene specifically induced by heat, NaCl, wounding and drought stress. Using GFP fusion vector, w123-smGFP was targeted both to nucleus. For the functional analysis of w123, His-tagged w123 recombinant protein was heterologously expressed in E. coli. The w123 recombinant cells showed enhanced heat tolerance compared to that of vector control cells. We suggest that dnaJ-like w123 protein function as molecular chaperone in the nucleus of the plant cell during various stresses.