Studies in hamster of peroxisomal events such as peroxisome proliferation (Lake et al., 1986; Gray et al., 1984), DNA synthesis (Lake et al., 1993), and induction of peroxisomal and mi- crosomal enzyme activity (Lake et al., 1986 and 1989; Sakuma et al., 1992) show that hamster is partially responsive to peroxisome proliferation. There has been no study regarding the in- ducibility of CYP4A gene(s) by peroxisome proliferators. Induction of CYP4A is an early marker of peroxisome proliferation and offers a more sensitive measure of responsiveness to peroxisome proliferation in hamster. RNase protection assays could be utilised to study the ex-
Figure 3.25 Peroxisome proliferation in rat primary hepatocytes. Isolated hepatocytes were seeded at a density of 2x106 cells per flask. Twenty-four hours after seeding cells were dosed with either 100 µM MCP (C, D) or 10µl DMSO (A, B) and cultured for a further 48 hours. Cells were then fixed and stained for peroxisomes by histochemical staining of peroxi- somal catalase as described in methods. Results are illustrated as viewed under low (A, C) and high (B, D) magnification of light microscopy. Magnification = x400 (A, C), x 800 (B, D).
A
B
pression and inducibility of CYP4A genes in hamster. As a prerequisite for such a study, cloning of CYP4A is required since no hamster CYP4A genes have been cloned. Once cloned (partial or full length cDNA), specific probes could be designed for RNase protection assay.
hmCypPR1
hmCypPR2
Figure 3.26 Design of PCR primers from alignment of CYP4A proteins. Forward (hmCypPR1) and reverse (hmCypPR2) primers for the cDNA cloning of putative hamster CYP4A genes were designed by aligning the amino acid se- quences of rat 4A1, 4A2 and 4A3 (P08516, P20816 and P24464 respectively), mouse 4a10 and 4a14, rabbit 4A6 (P14580) and human 4A11 (Q02928) using the pileup tool within the GCG sequence analysis program, and displayed using GeneDoc. The amino acid position of rabbit 4A6 are shown at the top of the alignment while the amino acid position on the left of the alignment corresponds to individual CYP4A genes. Black shading represent 100%, dark-grey represent 80% and light-grey represent 60% conservation of the aligned region. The regions of amino acid identity used to design PCR primers are underlined.
The CYP4A family has been identified in a number of species including human, mouse and guinea pig, and using this sequence information PCR primers were designed. By aligning the CYP4A proteins, primers CypPR1 and CypPR2 were designed from regions of high identity. Primers CypPR1 correspond to amino acid sequence 305-316 (925-959bp of CYP4A1 mRNA;
B A
Figure 3.27 cDNA (A) and amino acid (B) sequence of hamster CYP4A17. Three independent clones derived from PCR amlification of hamster liver cDNA using hmCypPR1 and hmCypPR2 primers were sequenced on both strands by oligo-primer walking. Full contig of individual clone was obtained using GELMERGE and GEL ASSEMBLE within GCG se- quence analysis program. Final consensus sequence was obtained by analysing the full sequence of the three clones using PILE UP, TED and GeneDoc programe. The deduced amino acid sequence was obtained from the cDNA sequence by using TRANSLATE within GCG program.
1 AAGCTTGGAT CCCATCTCAC TCATAGCAAA TTGTTTCCCA ATGCAGTTCC 51 TTGCTCCTCC CGAGAAGGGC AGGAATGAGT GGCTGTGTCG GGGAGAATCT 101 GCTGCAAACC TGGAAGGGTC AAACACCTCT GGGTTTGGCC ACACCTTCGG 151 GTTGTGGTGG AGTGCGTAAA TGGAGAGTAC AACTGTGGCA CCTTTGGGTA 201 AAGAGCGCCC ATCAGGGAAG GTGACAGGTG TGCTGAGCTC TCTGACAATG 251 GTTGGGGCAG GTGGGTAGAG CCTCAGGGAC TCCTTGATGC ACATGGTAGT 301 GTAGGGCATC TGGTCCAGGT GATTCCAGGT AATGGAGGAT CCATCTCCCA 351 GGAGGCTCTG AACTTCCTCC CTGCATCTCT GCCGGTATTC AGGGTGAGTG 401 GCCAGAGCAT AGAAGATCCA GGAGACTCCA CTGGCTGTGG TGTCATGTCC 451 CTCAAACATG AATGTGTCCA CCTCAGCACG TAGGTCCTTG TCAGACAAGA 501 ATTCAAGCTT
1 KLEFLSDKDL RAEVDTFMFE GHDTTASGVS WIFYALATHP EYRQRCREEV 51 QSLLGDGSSI TWNHLDQMPY TTMCIKESLR LYPPAPTIVR ELSTPVTFPD 101 GRSLPKGATV VLSIYALHHN PKVWPNPEVF DPSRFAADSP RHSHSFLPFS 151 GGARNCIGKQ FAMSEMGSKL
accession number M14972) at the 3’ end of the gene, while CypPR2 represents amino acid se- quence 437-446 (1381-1410bp) at the 5’ end of the rat CYP4A1 gene. Figure 3.26 shows the amino acid alignment of the regions of the CYP4A genes used to design the PCR primers.
First strand cDNA was synthesised from total hamster liver RNA using oligo-dT primers. When these cDNA products were subjected to PCR the amplification using CypPR1 and CypPR2 primers, an amplified fragment of the expected size, 500bp, was detected by agarose gel elec- trophoresis (Figure 3.28, lane 3 and 4). The same PCR reaction conditions also detected a 510bp fragment when rat CYP4A1 cDNA (in pSG5) was used as a template (Figure 3.28, lane 2) and this was absent in the negative control reaction with no template DNA added (lane 1). This
Figure 3.28 PCR of CYP4 genes from hamster liver. PCR reactions were performed using hmCypPR1 and hmCypPR2 primers on hamster liver cDNA, derived from MCP treated total RNA (lane 3-4). All PCR reactions include 1.5mM MgCl2 (lane 1, 2, 4) except lane 3 where 2.5mM MgCl2 was used. Lane 1 is negative control in which no template DNA was added. Lane 2 is amplification reaction using of pSG5-mPPAR-a plasmid DNA. Five ml of each reaction was analysed on a 1% agarose gel, run on 1xTBE at 90v for 90 minutes. Syber-Green was added to all samples prior to loading and the resolved DNA bands were visualised using a dark-reader. Molecular size of the DNA ladder are shown in base pairs.
100 200 300 400 500 1000 Ladder (bp) 1 2 3 4 1500
demonstrates that the amplified fragment was specific to the hamster cDNA template. The am- plified DNA fragment was purified from the reaction cocktail using a Qiaspin-quick column and subcloned into pGEM-T cloning vector. At least three independent clones containing the 500bp insert were purified and sequenced to obviate the artefacts arising from potential errors arising during the PCR process. Each clone was sequenced on both strands. Initial primers for sequencing were based on the T7 and SP6 promoter sequence of the pGEM-T vector, and the full-length sequence was obtained by oligo-walking process. The complete sequence of 510bp was analysed using the FASTA homology search in gene data bank (SRS) which revealed that the amplified fragment has high sequence similarity, but not identity to rat and mouse CYP4A genes (see later) and CYP4A genes from other species. Figure 3.27 shows the completed nucleic acid sequence and the deduced amino acid of this hamster CYP4A gene.