Cloning of DNA corresponding to rare transcripts of rat brain: evidence of transcriptional and post-transcriptional control and of the existence of nonpolyadenylated transcripts.

Abstract

To examine the expression of genes encoding rare transcripts in the rat brain, we have characterized genomic DNA clones corresponding to this class. In brain cells, as in all cell types, rare transcripts constitute the majority of different sequences transcribed. Moreover, when compared with other tissues or cultured cells, brain tissue may be expected to have an even larger set of rare transcripts, some of which could be restricted to subpopulations of neural cells. We have identified seven clones whose transcripts are nonabundant, averaging less than three copies per cell. Clone rgl3 (rat genomic 13) RNA was detected only in the brain, whereas RNA of a second clone, rg4O, was also detected in the brain and in a melanoma. Transcripts of rgl3 were found in cerebellum, cerebral cortex, and regions underlying the cortex, whereas rg4O transcripts were not detected in the cerebellum. Transcripts of both rgl3 and rg4O were found in pelleted polysomal RNA. RNA of another clone, rg34, was found in the brain, liver, and kidney but was found in pelleted polysomal RNA only in the brain, suggesting that its expression may be post-transcriptionally controlled. The remaining four clones represent rare transcripts that are common to the brain, liver, and kidney; rgl8 RNA is restricted to the nucleus, whereas rg3, rg26, and rg36 transcripts are found in the cytoplasm of all three tissues. Transcripts of the brain-specific clone, rgl3, and the commonly expressed clone, rg3, are nonpolyadenylated, presumably belonging to the high-complexity, nonpolyadenylated class of transcripts in the mammalian brain.The mammalian brain, the most complex organ in terms of function and structure, is also the most complex in terms of the diversity of its transcripts. The diversity or complexity of an RNA population can be measured by hybridizing RNA in excess to trace amounts of radioactive, nonrepetitive (unique-sequence) DNA, such that the amount of DNA driven into hybrids at saturation gives a direct measure of the percentage of nonrepetitive DNA transcribed. Complexity measurements have consistently shown that brain RNA in rodents displays a greater complexity than RNA from other tissues (mouse tissue [2,6,15,16,19,26,49]; rat tissue [8,9,13,14,17,25,28,43,47,51]). For example, Chikaraishi et al. (9) have found the nuclear RNA complexity of rat brain (31.2% of unique-sequence DNA coding capacity, assuming asymmetric transcription) to be higher than that of liver (21.8%) or kidney (10.6%).A higher proportion of nuclear RNA complexity (nearly two-thirds) is conserved as cytoplasmic or polysomal RNA in the brain than in the liver or kidney (3, 8)

Brain-specific polyA- transcripts are detected in polyA+ RNA: do complex polyA- brain RNAs really exist?

“…To date, there has been no report of a full-length polyAm clone for which the 3' and 5' ends have been determined. However, fragments of several brain-specific putative polyAm genes were identified from a genomic library of rat uniquesequence DNA (Brilliant et al, 1984). We show here that 2 different transcripts, typical of those previously termed polyA-mRNAs [brain specific, rare in abundance, postnatally expressed, and not retained on oligodeoxythymidine (oligo-dT) columns], are detected in low amounts as polyA+ RNA.…”

“…Preparation and labeling of clones rgl3, rglO0, TH, and NaCh. Clones rg13 and rglO0 are independent isolates of a library of phage M 13mp7 containing Sau3A DNA fragments from unique-sequence rat genomic DNA (Brilliant et al, 1984). The rg13 and rglO0 inserts were subcloned into the EcoRI site of the plasmid vector Sp65, which contains a promoter for Sp6 RNA polymerase.…”

Structure and Expression of the Guinea Pig Preproenkephalin Gene: Site-Specific Cleavage in the 3′ Untranslated Region Yields Truncated mRNA Transcripts in Specific Brain Regions

“…However, attempts to clone unique cDNAs from this class have met with little success. To date, only a few cDNA clones have been isolated which apparently correspond to unique, brain-specific, nonadenylated mRNAs (7,27). Other investigators were unable to identify cDNAs which were not represented in the poly(A) ϩ fraction of mRNA and have concluded that deadenylation in vivo, contamination of cytoplasmic mRNA with nuclear RNA, or nicking during RNA isolation were more likely origins of the observed complexity in the poly(A) Ϫ fraction (24,28,57).…”

Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene.

“…Total RNA from H35 cells or nuclear RNA from rat brain (containing a predominantly nondividing cell population) was prepared and dotted onto nitrocellulose. Figure 3 shows (9). BH.41+, BH.41-, SS1.6+, SS1.6-, SS1.1+, and SS1.1-were digested with HaeIII, end labeled, and hybridized with nuclear or total-cell RNAs from SD rat livers, H35 cells, and RT4-D1 cells.…”

“…In our laboratory, we have elaborated transcription mapping techniques to detect very low levels of RNA, i.e., on the order of 0.1 copy of RNA per cell (9). Using a modification of this technique in which single-stranded rDNA recombinants are cleaved with HaeIII, end labeled, and hybridized with total-cell and nuclear RNAs, we analyzed the region from + 125 in the rDNA precursor gene to the SstI site at -2.86 in the spacer.…”