Animal Treatment.

Two groups of animals (10 animals per group) were treated daily for 21 days with PLG (20 mg/kg, i.p.) or saline. Following a 2 day washout period, the animals were sacrificed, their brains removed and placed on ice for dissection. Various regions of the brain (striatum, pre-frontal cortex, medulla, cerebellum, hypothalamus, olfactory tubercle and hippocampus) are dissected out and immediately frozen on dry ice. The samples are stored at -80°C until needed.

RNA Preparation.

Total RNA from the striatum is isolated using the Tripure reagent method (Gibco Life Technologies, Gaithersburg, MD). The amount of mRNA obtained was determined by measuring the OD at 260 nm and the purity by the ratio of 260/280 nm. The integrity of the RNA (18S and 28S bands) was assessed by running 5 µg on a denaturing 1% agarose gel. Prior to use in RT-PCR, mRNA is purified using a DNase I (Gibco BRL Life Technologies) treatment to remove any contaminating chromosomal DNA. DNase treatment is done immediately prior to RT-PCR to ensure that the diluted mRNA is not degraded during storage.

Differential Display PCR.

Differential display RT-PCR (ddPCR) was performed using the GeneHunter (Nashville, TN) ddPCR RNAimage kit #1 (figure 1). Briefly, the purified mRNA undergoes three reverse transcription reactions with three different oligo-dT primers (H-T₁₁G, H-T₁₁C, H-T₁₁A). The products of each RT reaction are used in 8 different PCR reactions (MJ Research MiniCycler). Each PCR reaction uses the same oligo-dT primer used in RT and one of eight arbitrary primers. Hence, each mRNA sample undergoes a total of 24 PCR reactions.

Acrylamide Sequencing Gel.

For comparisons between control and PLG treated samples, the ddPCR process is conducted side by side on equal amounts of mRNA from both samples. Following the completion of the ddPCR reactions, the PCR products were separated by running the samples through a 6% denaturing acrylamide gel using a BioRad Sequi-Gen GT nucleic acid electrophoresis cell. Following electrophoresis, the gel is removed from the gel apparatus and dried using a BioRad gel slab drier. The dried gel is exposed to Kodak BioMax MR film for 3 days at room temperature. PCR products from similar reactions for the control and PLG samples are run in adjacent lanes for easy comparison of amplified bands. Those bands identified as being regulated by PLG were excised from the gel and solubilized in 100 µl dH₂O, precipitated and finally resuspended in 10 µl dH₂O.

cDNA Cloning and Sequencing.

The recovered cDNA (PCR fragments) is reamplified using the same primers by which they were generated. The resultant PCR products are run on a 1.7% agarose gel and their size is checked with what is expected from the acrylamide gel. The PCR generated cDNAs were cloned into the pCR-TRAP cloning system (GenHunter) using blunt end ligation and expressed in competent cells. Transformed cells were plated on LB plates containing 20 µg/ml tetracycline and incubated overnight at 37°C. The presence of cDNA inserts was screened for by using Tetracycline resistance (TetR) and checked by colony-PCR according to the pCR-TRAP protocol. The use of a phage Lambda repressor gene by this cloning system allows only those plasmids that have a cDNA insert to acquire TetR. A number of insert positive colonies were grown overnight in liquid culture (LB medium + 20 µg/ml tetracycline) and the plasmid DNA harvested the next day using QIAGEN miniprep system. Plasmid DNA was sequenced at the MOBIX facility at McMaster.

Northern hybridization.

The cDNA inserts were excised by Hind III restriction enzyme digestion and separated on a 1.7% agarose gel. The cDNA is recovered from the gel using the QIAEX II gel extraction kit (QIAGEN, Chatsworth, CA) and labelled using a random primed labelling kit (Gibco) and subsequently used as a probe in Northern hybridization. The cDNA probe is verified by Northern blot on both control and PLG treated RNA.

We implemented a strategy for identifying genes involved in the signal transduction pathway of the novel neuro-peptide PLG which employed the differential display of mRNA by RT-PCR technique. An overview of this technique is given in Figure 1.

Figure 1. Flow chart of differential display by RT-PCR technique.

Approximately 2400 genes were screened using the GeneHunter Kit, and we found three bands which were down-regulated due to treatment with PLG. Figures 2 and 3 are ddPCR gels which are representative of the results we obtained using this technique.

Figure 2. Representative sequencing gel of ddPCR fragments differentially regulated in striatal RNA samples from control and PLG treated rats. It can be seen that the indicated band is absent in the three PLG lanes.

Figure 3. Representative sequencing gel of ddPCR fragments differentially regulated in striatal RNA samples from control and PLG treated rats. It can be seen that the indicated band is absent in the three PLG lanes.

The bands were excised and the cDNAs were recovered and cloned into the pCR-Trap vector. Sequencing of the cDNAs revealed that some of the bands consisted of greater than 1 cDNA species. In total, 6 unique cDNAs were recovered; ranging in size from 133 bp to 228 bp (see table 1). Comparison of the nucleotide sequences we obtained with those in Genbank revealed that three of the cDNAs matched well with known genes (PRG1, PRG3 and PRG6), while three produced poor matches (PRG1, PRG3 and PRG6). The results of the Genbank search are summarized in Table 1.

----------------------------------------------------------------------------
cDNA  Length  Match Length  Blastn score/        NEAREST HOMOLOGY
       (bp)       (bp)      Expect Value   Gene sequence match (accession #)
----------------------------------------------------------------------------
PRG1    225   181/187;96%      323/7e-87   Homo sapiens full length insert
                                           cDNA clone ZD50B08 (AF086296)
PRG2    228    43/49;87%        50/1e-4   Genomic sequence from Mouse 11,
               33/36;91%                   complete sequence (AC002121)
               43/50;86%
               45/54;83%
PRG3    224   168/187;89%*     234/5e-60   Mus musculus mRNA for protein L,
                                           partial cds (AB009392)
PRG4    133    17/17;100%       34/4.8    Caenorhabditis elegans cosmid
                                           T01B7, complete sequence (Z66499)
PRG5    166    20/20;100%       40/0.1    Human Chromosome X, complete
                                           sequence (AC002418)
PRG6    166    88/97;90%       121/3e-26   Mus musculus major histocompatibility
                                           locus class II region (AF050157)
----------------------------------------------------------------------------

Northern blot analysis is used to confirm the expression of the differentially regulated cDNAs. Figure 4 is a northern blot which confirms the expression of PRG1 as well as comparison to the expression of ß-actin in the same samples.

Figure 4. Northern hybridization of probes for PRG1 and ß-actin to striatal RNA (20 µg) from control (lanes 1, 2 and 5) and PLG (lanes 2, 4 and 6) treated animals.

Figure 5 shows the expression of PRG1 as a percentage of ß-actin, and confirms that the expression of PRG1 was decreased in the striatum of animals treated with PLG.

Figure 5. Expression of PRG1 in control and PLG treated rat striatum.

Figure 6 is a northern blot using the cDNA PRG3 as a probe. The probe was hybridized against rat RNA (hypothalamus,hippocampus, pre-frontal cortex and cerebellum) as well as RNA from the human neuroblastoma SH-SY5Y cell line. The probe detected three bands in the rat RNA and two bands in the human RNA. This cDNA is known to match well with the sequences for the heterogeneous nuclear RNP protein L in the mouse and human, so it was expected to hybridize with both rat and human RNA. The human form of protein L RNA is 2033 bp in length while the mouse form is 958 bp in length. It is expected that the rat form of the RNA should be more similar to that of the mouse than of the human. Therefore, the band most likely to be that of protein L is band #3 in the rat and band #2 in the human RNA. Since protein L belongs to a large family of ribonuclear proteins, the other bands detected are likely to be other subtypes of ribonuclear proteins.

Figure 6. Northern hybridization of probe for PRG3 to RNA from rat hypothalamus (5 µg), hippocampus (17.6 µg), pre-frontal cortex (23.6 µg) and cerebellum (40 µg) (lanes 1, 2, 3 and 4 respectively) and human neuroblastoma cells (SH-SY5Y; 40 µg, lane 5).

Additionally, a Norther blot was performed using the cDNA PRG3 as a probe. This cDNA did not hybridize with RNA from rat hypothalamus, hippocampus, pre-frontal cortex cerebellum as well as SH-SY5Y RNA. This indicates that the corresponding RNA is either non-existent or expressed only in the striatum. Further testing will clarify this.

We have successfully used ddPCR to identify genes whose expression is regulated by the endogenous neuro-peptide PLG. Although none of the ddPCR fragments we obtained were exact matches with previously identified genes, three of the cDNAs displayed significant homology known gene sequences. This suggests that we have identified either rat homologs of genes cloned in other organisms, or additional members of a family of genes. We are currently in the process of confirming the expression of the cDNAs we have identified, and have obtained positive results for two of the three cDNAs tested to date. Subsequent experiments will involve full-length cloning of those cDNAs confirmed to be expressed in the striatum and regulated by PLG treatment. These experiments will allow us to characterize the role of the novel genes we have obtained herein in mediating the biological effects of PLG. This information will hopefully assist in elucidating the signal transduction mechanisms of PLG.

CONCLUSIONS.

* ddPCR experiments identified 6 unique cDNA species to be down-regulated by PLG treatment

* Sequencing revealed that all cDNAs were previously unidentified in rat

* Three of the cDNAs had poor homology with known genes

==> suggesting novel genes

* Three cDNAs had significant homology with previously identified genes from other species

==> suggesting rat homologs of genes

* Confirmation of expression by Northern hybridization has revealed that two of the three cDNAs tested to date are expressed in rat brain

* Expression of PRG1 has been confirmed to be down-regulated by PLG