Total RNA from post mortem human caudate nucleus, cerebellum, cerebral cortex and pheochromo- cytoma tissues has been prepared. Northern blot analysis ...
BJ!oscience Reports, Vol. 8, No. 3, 1988
A Highly Sensitive Northern Blot Assay Detects Multiple Proenkephalin A-Like mRNAs in Human Caudate Nucleus and Pheochromocytoma Hans-Jiirg Monstein ~,2. and Thomas Geijer 2 Received March 1, 1988 Total RNA from post mortem human caudate nucleus, cerebellum, cerebral cortex and pheochromocytoma tissues has been prepared. Northern blot analysis, using a single-stranded human proenkephalin A antisense probe (cRNA), revealed the existence of two different proenkephalin A-like sequences in the human caudate nucleus and pheochromocytoma RNA extracts of approximately 1400 and 1000 nucleotides in length respectively, whereas no specific RNA bands could be detected in the cortex and only the 1400 nucleotide band was present in the cerebellum. Under highly stringent hybridization conditions, the proenkephalin A-like RNA bands still appear, indicating that the detected RNA species have either identical or a closely related sequence to that of the well, characterized human proenkephalin A mRNA sequence. KEY WORDS: Northern blot, mRNA, proenkephalin A, cRNA probe, sensitivity.
The primary structure of the human proenkephalin A precursor protein has recently been elucidated by DNA-sequencing of the cloned proenkephalin A cDNA (1, 2). Progress in DNA recombinant technology has allowed the study of proenkephalin A m R N A distribution and its transcription in various tissues (2-5). Most commonly, RNA quantitation and distribution are studied either by Dot blot or Northern blot analysis, techniques that rely on the molecular hybridization technique, using the labelled cDNA probes. The most common uniform labelling methods are nick-translation, random-primer labelling and in vitro transcription of antisense RNA, the latter producing a high specific activity probe (for review see ref. 6). The use of uniformly labelled single stranded antisense RNA probes 5,2 Department of Microbiology, Biotechnology Group, Swedish University of Agricultural Sciences, Box 7025, S-750 07 UPPSALA, Sweden. 2 Department of Pharmacology, Uppsala University, BMC, Box 591, S-751 24 UPPSALA, Sweden. * Author for correspondence. 255
0144 8463/88/0600-0255506.00/0t~ 1988PlenumPublishingCorporation
Monstein and Geijer
(cRNA) allows hybridization under more stringent conditions due to the high stability of R N A : R N A hybrids as compared to D N A : D N A or RNA :DNA hybrids. In this study, we show that stringent hybridization conditions with a high specific activity proenkephalin A cRNA probe, detects two proenkephalin A-like RNAs in human caudate nucleus and human phenochromocytoma.
MATERIALS AND METHODS Preparation of RNA Total RNA was extracted from human caudate nucleus, cerebellum and cerebral cortex respectively (less than 24 h post mortem) as described earlier (7). RNA from human pheochromocytoma which was obtained by surgery was prepared according to the procedure of Chomczynski et al. (8). The RNA preparations were routinely checked for degradation on an agarose minigel.
Northern Blots Northern blots were performed as described earlier (7); in brief, 10/~g of total RNA was separated on a 1.2% agarose formaldehyde gel, followed by transfer to Hybond-N membranes (Amersham) in 25 mM Na-phosphate buffer, pH 6.4. Filters were baked at 80~ for 2 hours.
Hybridization Hybridization was performed as described by Maniatis et al. (9). The filters were first prehybridized in 10ml of a solution containing 50% formamide, 0.6MNaCI, 120mM Tris-HClpHT.5, 8mM EDTA, 1% SDS and 1% non-fat dried milk powder (10) at 60~ in a sealed plastic bag in a waterbath for at least 4 hrs. After prehybridization, the nylon membranes were incubated at 60~ for 20 to 24 hrs in the hybridization buffer described above. Hybridization was carried out with a SP-6 derived single-stranded "complementary" human proenkephalin A cRNA probe (7).
Posthybridization of the Nylon Membranes Following hybridization, nylon membranes were washed 3 • 15min in 0.1 • SSC (15 mM NaC1, 1.5 mM Na-citrate), 0.1% SDS, 0.1% Na-pyrophosphate under different stringent conditions (Text in Fig. 1 A - C ) and air dried at room temperature. Bands were visualized by exposure for 1 to 2 days at -70~ to Kodak X-O mat film, using a DuPont intensifier screen.
Construction of the Riboprobe pSP65-5 Plasmid The RI-SalI fragment, containing the entire human proenkephalin A DNA-sequence, was cut from plasmid pCD-5 and subcloned into the promega
Pre, enkephalin A-like mRNAs
vector pSP-65 (11). The recombinant plasmid pSP65-5 was cloned and propagated in E. coli HB 101 cells, pSP65-5 was linearized with endonuclease Puv II prior to transcription, using SP-6 RNA polymerase and Promega's Riboprobe-kit according to the manufacturer's instructions. In vitro transcribed [32P]a~UPT-labelled cRNA is 800 nucleotides in length and was heat denatured in a boiling waterbath four 1 to 2 rain prior to hybridization.
RESULTS AND DISCUSSION
Total RNA extracted from human post mortem caudate nucleus, cerebellum, cerebral cortex and human pheochromocytoma tissues was examined for the presence of proenkephalin A RNA by Northern blot analysis. As a hybridization probe [32p]o;UTP-labelled proenkephalin A antisense RNA was used. This cRNA was transcribed from a cDNA encoding proenkephalin A, cloned in plasmid pSP65 which contains the SP6 RNA-polymerase initiation site upstream to the antisense cloned DNA. Hybridization was carried out in 50% formamide at 60~ however Denhardts solution was replaced by non-fat dried milk powder (10). This procedure not only simplified the hybridization procedure but also yielded an increased signal to background ratio. Due to the thermal stability of R N A : R N A hybrids, Northern analysis was performed under more stringent conditions than usually applied with nick-translated or random primed DNA probes. However, we often observed a high background hybridization mainly to 18S and 28S rRNA respectively, which often could not be removed by recommended washing procedures. If Hybond-N filters were washed at 60~ for 3 x 15rain in 0.1 x SSC, 0.1% SDS and 0.1 Na-pyrophosphate, multiple bands became visible after exposure to X-ray film. Fig. 1 A, lane 1, shows the high
Fig. 1. Northern blot analysis of total RNA from human caudate nucleus (lane 2), cerebellum (lane 3) and cerebral cortex (lane 4), As a control, a poly A ( - ) R N A fraction from A549 cells was included (lane 1). 10 ~g total RNA were electrophoretically separated on a 1.2% denaturating agarose gel, transferred to Amersham Hybond-N-membrane and hybridized with a [32p]o:UTP-labelled human proenkephalin A cRNA probe as described in Materials and Methods. After hybridization, the filter was initially washed in 0.1 • SSC, 0.1% SDS, 0.1% Na pyrophosphate at 60~ for 3 x 15 min under moderate agitation in a water bath (A) air dried and exposed to Kodak X-Omat film. Subsequently, the washing procedure was repeated twice under conditions of increased stringency such as 65~ (B) and 75~ (C), respectively. 28S and 18S indicate the positions of ribosomal RNA, and HE the position of the human proenkephalin A-like RNAs.
Monstein and Geijer
background hybridization to 28S rRNA as well as in some extent to 18S rRNA and to a high molecular weight band, presumably the 45S rRNA precursor. High background hybridization also occurred in caudate nucleus (lane 2), cerebellum (lane 3) and cortex (lane 4) RNA extracts. Under the hybridization conditions described, proenkephalin A-like RNA bands can be distinguished from the relative high background hybridization only in the caudate nucleus preparation. Subsequently, the same filter was subjected to more stringent washing conditions. After the membranes had been washed at 65~ in the same buffer as mentioned before, it was found that proenkephalin A-like RNA was present in both caudate nucleus and cerebellum extracts (Fig. 1B, lanes 2, 3) whereas no RNA-band could be detected in cortex (Fig. 1B, lane 4). The poly A(-) fraction from A549 cells showed only a weak band at the 28S rRNA position (Fig. 1A, lane 1). A further increase of the filter washing temperature to 75~ completely reduced background hybridization, both to the filter and to unspecific RNA such as ribosomal RNA. With the highly stringent conditions used, the caudate nucleus RNA extracts reveal two distinct RNA bands of approximately 1400 and 1000 nucleotides in length, and both RNAs seem to be expressed at a much higher
Fig. 2. Northern blot analysis of 10/~g total RNA from human caudate nucleus (CN), cerebellum (CE) and cortex (CO). Arrows indicate the positions of 28S and 18S rRNA, in vitro [32p]o(UTPRNA control transcripts of 1386, 557 and 225 nucleotides in length, respectively (Promega, Riboprobe). HE marks the positions of the approximately 1400 and 1000 nucleotide long human enkephalin-A like RNAs as described in the text.
Proenkephalin A-like mRNAs
level than in cerebellum, where only the 1400 nucleotide species is present. In a separate experiment, 10/~g total R N A from caudate nucleus, cerebellum and cortex was electrophoresed in parallel with three high specific activity labelled [32p]o~UTP-riboprobe R N A markers of 1386, 557 and 225 nucleotides in length. Analysis of this Northern blot indicated that the two proenkephalin A-like RNAs in caudate nucleus are 1400 and 1000 nucleotides, whereas the cerebellum species is 1400 nucleotides (Fig. 2). To confirm the identity of the hybridization signals, 10/~g total R N A from a A549 poly A(-)fraction, the neuroblastoma S K - N - M C and SH-SY5Y cell lines, human CLL-leukocytes, small cell lung carcinoma cells, and from human pheochromocytoma (Fig. 3, lanes 1-6), were subjected to Northern blot analysis. Hybridization and subsequent wash of the filter at 65~ (Fig. 3A) and 75~ (Fig. 3B) respectively showed a strong single band of 1400 nucleotides in the S K - N - M C RNA extract. However, in the pheochromocytoma RNA extract the same pattern was observed as in the human caudate nucleus. In the present study, RNA blot hybridization analysis using a high specific activity [3Zp]o:UTP-labelled proenkephalin A antisense RNA demonstrated that the human caudate nucleus and a pheochromocytoma express two proenkephalin A-like RNAs of distinct size, estimated to be 1400 and 1000 nucleotides in length. cDNA sequence analysis has earlier revealed that human proenkephalin A m R N A is approximately 1400 nucleotides in length (1, 2). In an earlier study (7)
Fig, 3. Northern blot analysis of proenkephalin A expression. 10/zg total RNA from human neuroblastoma cell lines S K - N - M C (lane 2) SH-SY5Y (lane 3), human CLL-leukocytes (lane 4), small cell lung carcinoma U-1285 (lane 5) and human pheochromocytoma (lane 6) were separated on a denaturating agarose gel and transferred to Hybond-N membrane (Amersham). 10/~g of poly A(-)RNA from A549 cells was included as a control (lane 1). Hybridization was carried out with a 800 nucleotide long human proenkephalin cRNA as described in Materials and Methods. Initially, the filter was washed in 0.1 x SSC, 0.1% SDS, 0.1% Na-pyrophosphate at 65~ for 3 • 15 min (A) under moderate agitation in water bath, exposed to a Kodak X-Omat film at -70~ for 2 days, followed by a further filter wash at 75~ for 3 • 15 min (B). HE indicates the position of the proenkephalin A-like sequences.
Monstein and Geijer
we described the existence of two distinct proenkephalin A-like RNAs of similar size to be present in human leukocytes from patients with chronic lymphocytic leukemia (CLL). Kilpatrik et al. have shown that rat ovaries express a 1400 nucleotide and a smaller proenkephalin A species, whereas in the rat testis a 1400 and a 1900 nucleotide long proenkephalin A-like RNA was found (12, 13). Also, Yoshikawa et al. (14) reported the presence of two proenkephalin A-like RNAs in rat striatum. In addition, proopiomelanocort in mRNA heterogeneity has also been reported by Civelli et al. (15) who found that POMC-like RNA from rat amygdala and cerebral cortex migrated differently on a denaturating agarose gel as compared to POMC-like RNA from the hypothalamus. An explanation for the molecular weight heterogeneity of proenkephalin A-like RNA in human caudate nucleus, pheochromocytoma and CLL-cells could be that the different structures may be generated by post-transcriptional mechanisms, such as selection of different poly A-addition sites. Oligo d(T) column chromatography implies that both RNAs are polyadenylated (Fig. 4). Inspection of the primary DNA sequence of the proenkephalin A gene reveals that there are several potential polyadenylation sites (16). A polyadenylation signal-sequence T A A T A T C is found at the immediate boundary of the translatednontranslated region in the proenkephalin A cDNA, where the T A A trinucleotide comprises the stop condon. The T A A T A T C sequence has structural similarities with the polyadenylation consensus-signal A A T A A A (17), which in the proenkephalin A gene is found approximately 250 nucleotides distal to the stop codon and gives rise to the well-characterized 1400-nucleotide species.
Fig. 4. Northern blot analysis of total RNA from human caudate nucleus (lane 1) and poly A § selected RNA (lane 2). Hybridization was as described in Fig. 3. Arrows indicate the position of the proenkephalin A-like RNAs (HE).
One could also argue that processing of the 1400 nucleotide specie into its smaller counterpart by a tissue specific RNase could be another explanation for the observed RNA pattern. The existence of such an RNase has been implied to be present in rat brain tissues by Civelli et al. (15). It is of course also possible that the observed RNA pattern results from unspecific degradation. However, under the stringent hybridization conditions used in this study, randomly degraded RNA would rather appear as a smear than two distinct bands. In summary, the application of highly stringent hybridization conditions, in combination with a high specific activity labelled antisense (cRNA) probe, allows
Proenkephalin A-like mRNAs
the detection of low abundant RNAs in total R N A extracts. The results obtained also indicate that proenkephalin A-like RNAs of different sizes may be present in human caudate nucleus and pheochromocytoma tissues.
ACKNOWLEDGEMENTS This work was supported by the Department of Microbiology and by the Bank of Sweden Tercentenary Foundation. We thank Professor Lars Terenius for critical reading and comments on the manuscript.
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