Abstract
We have studied the expression and regulation of the rat testis cytosolic aspartate aminotransferase gene. The cytosolic aspartate aminotransferase activity was 5-fold lower in the testis than in the liver and kidney. A 1.9-kilobase mRNA form was detected in the rat testis in contrast to the 2.1- and 1.8-kilobase forms present in other organs. Using Northern blot and S1 mapping analyses, we found that the proximal polyadenylation site was almost exclusively used in the testis as opposed to other organs where the distal site was preferentially used. RNase protection and primer extension analysis showed that transcription was initiated at multiple sites in all organs, but the pattern of those start sites was different in the testis; in particular, a novel transcription start site was specifically detected in this organ (at position -115 from the translation start site). This site was first observed in 29-day-old rats and was maximally utilized in the adult testis. DNase I footprinting using testis nuclear extracts revealed the presence of three sites of DNA-protein interaction in the 250-base pair proximal promoter, a pattern similar to the one found using liver nuclear extracts. However, the proteins bound had different properties as shown by gel retardation experiments. We conclude that the pattern of transcription initiation and the polyadenylation site selection of a housekeeping gene can be tissue-specific.
Highlights
We have studied the expression and regulation of thhoeusekeeping gene promoters presumably leads toa cluster of rat testiscytosolicaspartateaminotransferasegene
1.9-kilobasemRNA form wasdetected in the rat testis ipnlay specific properties that areatypical of housekeeping gene contrast to the 2.1- and 1.8-kilobase forms present in otherorgans.UsingNorthernblotandS1mapping promoters
The expression of the cAspAT housekeeping gene was examined in several tissues including the testis
Summary
RNA Preparation-Total RNA was isolated from different tissues usingtheguanidiumthiocyanateextractionmethoddescribed by Chomczynskiand Sacchi (14).Briefly, 1g of tissue was homogenized in. Northern H o t Hybridization-Poly(AY RNA (2-5 pg) was electrophoresed on a horizontal 1.2%agarose, 2.2 M formaldehyde gel (17)and. The reaction was stoppbeydaddition of 80 pl of a solution containing M4ammonium acetate, 2m0 ~ EDTA, pH 8, and FIG.[1].A, Northern blot analysis of 2 pgof poly(A)+RNAfrom rat liver. Primer Extension Analysis-Oligonucleotides were 5' end-labeled, using [ Y - ~ ~ P I ~ (A30T0P0 CUmmol, Amersham Corp.) and T4polynucleotide kinase tao specific activity of 6 x lo6cpdpmol(19).Poly(AY RNA (10 pg) were added to 1.x6 lo6cpm of labeled oligonucleotide, and the mixture was ethanol-precipitated. Following a n incu- Gel Retardation Assay-Probes were double-stranded oligonucleotibation at 42 "C for 90 min, the reaction was stoppedby addition of 20 des labeled using the Klenow fragment of DNA polymerase I (8).
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