Identification of the cyclin D1b mRNA variant in mouse

Abstract

Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and over-expressed in many cancers. The cyclin D1b mRNA variant was established in human cells and recent functional analyses revealed that its protein product harbors unique activities in human cancer cells. By performing reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) experiments, we identified the cyclin D1b mRNA variant in mouse. Similar to its human counterpart, the mouse cyclin D1b transcript consists of exon 1, 2, 3, 4 and part of intron 4, and contains a long open reading frame (ORF). The predicted peptide from this ORF is 34-amino acid longer than the human cyclin D1b. The expression of this mouse mRNA variant was investigated. It appears to be expressed ubiquitously and differentially in various mouse cell lines and tissues and its level might be proportional to that of the canonical endogenous cyclin D1a mRNA.

Fig. 1

(a) RT-PCR amplification with the cyclin D1 primer pair D1-L723 and D1INTRON4-R246. The left hand primer anneals to exon 3 sequence, and the right hand primer anneals to sequence in intron 4. The expected size of the PCR product is 498 bp. When genomic DNA (gDNA) was used as template, 200 ng from each sample was used in PCR. Lane 1–4, gDNA from mouse pancreatic tumor tissue, tail tissue, NH2 cells and M4 cells, respectively; lane 5, no template; lane 6–10, cDNA from cells 67NR, 66cl4, 168FARN, 4T1, and 4TO7, respectively; lane 11–14, cDNA from NH2, NN2, M4 and M8 cells, respectively; lane 15, 1kb⁺ DNA ladder from Invitrogen. (b) RT-PCR amplification with the cyclin D1 primer pair D1-L228 and D1INTRON4-R246. The left hand primer is a few base pairs upstream of the cyclin D1 protein translation start codon ATG in exon 1, and the right hand primer is in intron 4. The expected size of the PCR product is 974 bp. Lane 1–2, gDNA from NH2 and M4 cells, respectively; lane 3, no template; lane 4–5, cDNA from NH2 and M4 cells, respectively; lane 6, 1kb⁺ DNA ladder. (c) Rapid amplification of cDNA ends (RACE). 5′ RACE and 3′ RACE were performed as described in Materials and Methods. The template used in the PCR amplification is cDNA prepared from NH2 cells. In order to improve the chance of getting more specific amplification, we reproduced the reaction at 4 separate annealing temperatures: 57, 60, 62, and 65°C. (d) Comparison of cyclin D1a and cyclin D1b mRNA levels in NN2 and NH2 cells, and M8and H5 cells by RT-PCR. Cyclin D1a specific primer pair is D1-L723 and D1-R1064; cyclin D1b specific primer pair is D1-L723 and D1INTRON4-R246 (Fig. 2a). PCR amplification of β-actin served as cDNA template loading control. PCR products were separated by agarose gel electrophoresis (1% agarose in 0.5× TBE buffer). Each experiment was repeated a minimum of three times

Fig. 2

(a) Illustration of the locations of primers used in PCR amplifications and the components of mouse cyclin D1a and cyclin D1b cDNA. Coding exon sequences are represented by black rectangles; non-coding exon sequences are represented by empty rectangles; and cyclin D1b cDNA fragment derived from intron 4 are represented by a shadowed rectangle. (b) Comparison of the cyclin D1b peptide sequences derived from human and mouse intron 4, respectively