Identifying Genes Potentially Regulated in the Hippocampus by DNA Recombination
Jessenia Yaris Laguna Torres1, Edgardo Castro1, Yarelis Soto1, Juan Fernández2, Yamila Goenaga1, Iván Santos1, Humberto Ortiz Zuazaga2 and Sandra Peña de Ortiz1
1Departments of Biology and 2Computer Science , University of Puerto Rico, San Juan, PR, USA
We assess here the importance of genomic diversification mechanisms in consolidation of hippocampal dependent learning. Our overarching hypothesis is that long-term memory formation involves a regulatory mechanism of DNA recombination in the hippocampus that includes the activation of effector molecules, such as DNA ligases, and the rearrangement of specific target genes. Thus, using context fear conditioning as a model of learning in C57BL/6 mice, our main goal is to study genetic changes associated to DNA rearrangement and their significance in hippocampal dependent memory formation. Our previous studies using pharmacological blockade of DNA ligase function, showed that this treatment specifically blocked consolidation of context fear conditioning without interfering short-term memory or reconsolidation (Colón-Cesario et al., 2006). Present studies have determined that similar treatments block remote memory of context fear conditioning and impair hippocampal synaptogenesis. These studies are being followed up using DNA ligase IV knockdown approaches in the hippocampus as well as gene microarray analysis to identify the target genes for this mechanism of gene regulation in memory formation. In conclusion, context fear conditioning results in associative learning-specific changes in hippocampal gene expression, some of which may be associated to genomic diversification processes in the brain and memory consolidation.
Supported by: NIH
*S.P.O. grants SOGGMO 8102-26S1, 5P20-RR015565, 2 R25 GM61151
*MARC undergraduate fellowship to Jessenia Yaris Laguna Torres
*BIOMINDS undergraduate fellowship to Jessenia Yaris Laguna Torres
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Methodology
Polymerase chain reaction (PCR) is a method that allows exponential amplification of short DNA sequences (usually 100 to 600 bases) within a longer double stranded DNA molecule. PCR entails the use of a pair of primers, each about 20 nucleotides in length, that are complementary to a defined sequence on each of the two strands of the DNA. These primers are extended by a DNA polymerase so that a copy is made of the designated sequence. After making this copy, the same primers can be used again, not only to make another copy of the input DNA strand but also of the short copy made in the first round of synthesis. This leads to logarithmic amplification. Since it is necessary to raise the temperature to separate the two strands of the double strand DNA in each round of the amplification process, a major step forward was the discovery of a thermo-stable DNA polymerase (Taq polymerase) that was isolated from Thermus aquaticus, a bacterium that grows in hot pools; as a result it is not necessary to add new polymerase in every round of amplification. After several (often about 40) rounds of amplification, the PCR product is analyzed on an agarose gel and is abundant enough to be detected with an ethidium bromide stain. For reasons that will be outlined below, this method of analysis is at best semi-quantitative and, in many cases, the amount of product is not related to the amount of input DNA making this type of PCR a qualitative tool for detecting the presence or absence of a particular DNA. In order to measure messenger RNA (mRNA), the method was extended using reverse transcriptase to convert mRNA into complementary DNA (cDNA) which was then amplified by PCR and, again analyzed by agarose gel electrophoresis. In many cases this method has been used to measure the levels of a particular mRNA under different conditions but the method is actually even less quantitative than PCR of DNA because of the extra reverse transcriptase step. Reverse transcriptase-PCR analysis of mRNA is often referred to as “RT-PCR” which is unfortunate as it can be confused with “real time-PCR”. (Dr. Hunt, 2007)
Polymerase Chain Reaction
Previous Findings
