Laura F. Newell and Dr. Randy L. Bennett, Zoology
Recently, considerable emphasis has been placed on the similarity, or homology, of genes in different organisms. By comparing gene sequences, one can see that a large proportion of genes in a fly, for example, has unequivocal homologues in vertebrates, and vice versa. Homologies have been found in many genes involved in the development of all organisms, including the homeotic genes, a class of developmental genes which are conserved throughout the animal kingdom.
Our understanding of homeotic genes and their role in development has been significantly enhanced by molecular and genetic analysis of Drosophila melanogaster, the fruit fly. However, the analysis of other organisms is necessary in order to allow large comparisons to be made among all animals.
One of Drosophila’s most studied homeotic genes is the Ultrabithorax, or Ubx gene. It is essential for the correct patterning of the anterior abdomen and posterior thorax (1). Tribolium castaneum, the red flour beetle, is an organism found to contain many homologs to Drosophila developmental genes. Of particular interest is the Ubx homolog in Tribolium, which is the Ultrathorax, or Utx gene. The Utx gene is important in the regulation of transcription and of other proteins that regulate transcription.
The goal of my research project was to complete the cloning of the Utx gene of Tribolium castaneum. Although many portions had already been cloned, the gene had not yet been cloned in its entirety. Of the gene’s total length of 2.5 kD, approximately 2.0 kD had previously been cloned.
The first step in the cloning process was to prepare RNA from Tribolium embryos at a stage in development at which it was known that Utx was expressed. The embryos were washed in a 50:50 bleach and water solution, rinsed, and then ground under liquid nitrogen. The total RNA was isolated from other cellular material using a Guanidinium/Cesium protocol. The result was a small pellet of RNA that was subsequently resuspended in water and stored at –80°C.
Next, a complementary DNA copy, or cDNA, of the RNA molecule was made. The cDNA was modified in a manner that allowed for the screening of a DNA library using polymerase chain reaction (PCR). This process was completed using the Clontech Marathon Kit, designed for the production and amplification of cDNA (2).
To construct the complementary DNA, a cDNA primer was used to initiate transcription of the RNA sample. The reaction was catalyzed by the enzyme reverse transcriptase, which synthesized a DNA chain on an RNA template. DNA polymerase was then used to convert the single-stranded DNA molecule into double-stranded DNA.
Next, the library of adaptor-ligated double-stranded cDNA was created. DNA “adaptors” were linked to the cDNAs in the library, which was then screened using polymerase chain reaction. Primers recognizing the 3′ and 5′ ends of the cDNA (from the adaptor ligation step) were used in combination with Utx-specific primers designed from the previously cloned portions of the Utx gene. This process allowed the amplification of the Utx clones. We were able to obtain a high level of specificity for the Utx gene, since the Utx primers were only able to hybridize with the Utx clones. The process of PCR worked like a copy machine, generating a large number of copies of the desired DNA sequences. The PCR products were submitted to gel electrophoresis, in which samples were loaded into small wells in an agarose gel and exposed to an electric current. The resulting bands were studied to assess the size and purity of the DNA fragments from the PCR reactions. The genuine products were cloned into bacterial plasmids.
Although we were able to obtain PCR products of the entire Utx gene, not all of the products were cloned into a bacterial plasmid due to time constraints. More time than initially anticipated was needed to clone all of the gene. Numerous attempts, using different combinations of Utx-specific primers and 5′ and 3′ primers, were required. However, I was able to finish the critical steps of the project, and the completed clones can be used to continue the analysis of the Utx gene.
References
- Lewis, E. 1978. Nature 276: 141-152.
- Clontech. 1998. Clontech Laboratories Inc.