Ghazala Hashmi

Injection of DNA into plant and animal tissues with micromechanical piercing structures

Silicon micromachining has been used to fabricate microprobes for injecting DNA into cells. Arrays of very sharp pyramidal points are etched on a silicon substrate. Pressing these points into a culture of cells, allows biologically active material to cross the cell wall barrier. Using the microprobes, DNA has been injected into plant (tobacco leaves) and animal (nematodes) cells.

Thermal Response of Heterorhabditis bacteriophora Transformed With the Caenorhabditis elegans hsp70 Encoding Gene

A heat-shock response is induced when cells are exposed to temperatures slightly higher than their optimal physiological temperature. This response is based on the synthesis of heat-shock proteins encoded by the heat-shock genes. A correlation between the increased thermotolerance and production of 70-kDa heat-shock protein (hsp70) has been observed in many organisms. We tested this hypothesis by transferring a Caenorhabditis elegans heat-inducible hsp70 A-encoding gene into the entomopathogenic nematodes Heterorhabditis bacteriophora Hp88. Successful transformation of the gene was confirmed by Southern blot hybridization and polymerase chain reaction. Our blot studies showed that the transgenic nematodes contained five to ten copies per genome of the introduced hsp70 A gene. hsp70 mRNA transcripts were detected in both wild-type and transgenic nematodes. Transcripts increased severalfold in transgenic nematodes upon heat shock. Infective juveniles of both transgenic and wild-type nematodes that exposed to a sublethal heat treatment (35 degrees C) for 2 h followed by a normally lethal heat treatment (40 degrees C) for 1 h. More than 90% of transgenic nematodes survived heat treatment, compared to 2% to 3% of the wild-type strain. Our observations establish that overexpression of hsp70 A gene resulted an enhanced thermotolerance in the transgenic nematodes. The transgenic nematodes displayed normal growth and development.

Polymorphism in heat shock protein gene (hsp70) in entomopathogenic nematodes (rhabditida)

Abstract 1. 1. All organisms tested respond to a sudden increase of temperature by synthesizing heat-shock proteins, which helps organisms to survive high temperature. A correlation with increased thermotolerance and production of major 70 kDa protein has been observed in many organisms. 2. 2. Many studies have been designed on a large number of animal species to assess their adaptation to different thermal environments. Genetic analysis of the hsp 70 gene in entomopathogenic nematodes inhabiting different environments may provide insight into the physiological roles of hsps in these nematodes and could be useful for ecological studies. 3. 3. To assess variation among species of entomopathogenic nematodes for thermotolerance, we initiated a search for the molecular organization of heat-inducible hsp 70 genes in these nematodes. Five Heterorhabditis species/isolates with different temperature optima for survival and one warm-adapted species of Steinernema were tested 4. 4. PCR and RFLP analyses of hsp 70 in Heterorhabditis species and S. scapterisci demonstrated a putative homology with the Caenorhabditis elegans hsp 70 A gene, thus indicating evolutionary conserved nature among different nematode species. 5. 5. RFLPs with the hsp 70 A gene probe revealed different banding patterns for Heterorhabditis species and isolates. 6. 6. This is the first report on the identification of any hsp 70 gene in entomopathogenic nematodes. 7. 7. Our observation establishes a unique correlation between geographical distribution and polymorphisms for hsp 70 A gene in these nematodes.

Transformation of Nematodes by Microinjection

The free-living nematode, Caenorhabditis elegans, has been an important model system in the study of developmental and cell biology. Significant advances in mapping and sequencing the C. elegans genome have aided our understanding of fundamental biological processes. Development of an efficient method for gene transfer has been a key tool accelerating C. elegans research advances (Kimble et al., 1982; Stinchcomb et al., 1985., Fire, 1986; Mello et al, 1991). Integrative transformation in C. elegans is reproducibly achieved after microinjecting DNA directly into maturing oocyte nuclei (Fire, 1986). Heritable extrachromosomal DNA transformation in C. elegans was first described by Stinchcomb et al. (1985) after microinjecting DNA into the gonad cytoplasm. DNA molecules injected into the cytoplasm of the C. elegans hermaphrodite gonad undergo a transient period of reactivity. This results in the formation of large heritable extrachromosomal structures that experience very little further rearrangement (Mello et al., 1991). Germ cell nuclei in C. elegans develop initially in a syncytium (a multinucleate mass of protoplasm resulting from fusion of cells) and when cell membranes later envelop them, the exogenously added DNA is packaged into the oocyte. Transforming DNA is generally not integrated into the chromosomes, but rather is maintained as a concatamer (i.e. unite in a chain) of introduced sequences.