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. 2001 Dec;75(23):11907-12.
doi: 10.1128/JVI.75.23.11907-11912.2001.

Murine leukemia virus proviral insertions between the N-ras and unr genes in B-cell lymphoma DNA affect the expression of N-ras only

J Martín-Hernández  1 , A B Sørensen, F S Pedersen
Affiliations

Murine leukemia virus proviral insertions between the N-ras and unr genes in B-cell lymphoma DNA affect the expression of N-ras only

J Martín-Hernández et al. J Virol. 2001 Dec.

Abstract

Akv1-99, a variant of Akv murine leukemia virus, induces B-cell lymphomas with nearly 100% incidence and a mean latency period of 12 months after injection into newborn NMRI mice. PCR amplification and sequence analyses of DNA flanking integrated proviruses revealed proviral insertion into the N-ras/unr (upstream of N-ras) locus in 2 out of 13 B-cell lymphomas, both of which appeared clonal by Southern blotting analysis. These two tumors showed increased expression levels of N-ras by Northern blotting, as did a third tumor shown by reverse transcriptase PCR to have a nonclonal provirus integration located in the same area. However, no significant changes in expression were observed when using a specific probe for the unr gene. All proviruses were integrated in the same transcriptional orientation as unr and N-ras genes. By promoter insertion, the two Akv1-99 proviruses integrated between exon -1 and exon 1 of N-ras gave rise to two different spliced products, whereas the provirus integrated into unr used only an exon skipping pattern. The absence of mutations of the N-ras codons 12, 13, 18, and 61 suggests that activation of the proto-oncogene is exclusively due to overexpression by retroviral promoter insertion, and furthermore, Northern blot analyses indicate that the expression of unr is unaffected by N-ras overexpression even in the case where the unr gene itself is the target of proviral insertion. Thus, altogether our findings indicate that overexpression of N-ras plays a role in development of murine leukemia virus-induced B-cell lymphomas, leaving the expression of the tightly linked unr gene unaltered.

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Figures

FIG. 1
FIG. 1
Diagram showing the locations and orientations of the three Akv1-99 insertions detected in tumor 3 (before nucleotide 335), tumor 9 (before nucleotide 1062), and tumor 11 (before nucleotide 1085) in this study (gray arrowheads) as well as previously reported insertions, SL3-3 (before nucleotide 668) and VST27016G1 (before nucleotide 1084) (11, 25) (black and white arrowheads, respectively). All five proviruses are inserted within an area of about 800 bp. Arrows indicate orientations of transcription of unr and N-ras. Shown below are the probes made by PCR that were used for Southern and Northern hybridizations. Probe A (915 bp) covers the very last untranslated 3′ region of unr and the first two N-ras exons (positions 382 to 1297); probe B (646 bp) comprises the first two N-ras exons (positions 651 to 1297); probe C (369 bp) contains the last 3′ exon of unr (positions 1 to 369). Positions indicated correspond to the GenBank sequence with accession no. L19607. ut, untranslated.
FIG. 2
FIG. 2
Southern blot analysis of HindIII-digested tumor DNAs from Akv1-99 tumors hybridized with a mouse N-ras probe (probe A), shown in Fig. 1. A rearranged fragment of about 17 kb in tumors 3 and 11 can be detected, while no arrangements are seen in the remaining tumors. Arrowheads indicate positions of the germ line (8 kb) and rearranged fragments (17 kb).
FIG. 3
FIG. 3
Northern blot hybridizations of RNA (23 μg) extracted from 11 independent tumors induced by Akv1-99. Hybridization was performed using either probe A (A), probe B (B), or probe C (C) (probes are shown in Fig. 1). Arrowheads indicate N-ras mRNA sizes. Tumor numbers are given on top of each lane. Hybridization with glyceraldehyde-3-phosphate dehydrogenase was used as an internal control (D).
FIG. 4
FIG. 4
RT-PCR analyses of tumor 3. (A) Representation of the provirus integrated into the unr gene. Primer c or d (positions 217 to 242 or 269 to 290, respectively; positions correspond to the GenBank sequence with the accession no. L19607), together with primer e, was used for the amplification of possible unr transcripts. Primer e hybridizes to the 5′ end of a poly(T) primer [5′-GGGTCTAGAGCTCGAGTCAC(T)16V-3′ (V = A/G/C)] which was used for reverse transcription. The figure is not drawn to scale. ut, untranslated; LTR, long terminal repeat. (B) PCR amplifications of the reverse-transcribed cDNA. Amplifications were done twice on the same preparation of cDNA. Primers used are indicated on top of each lane. Sizes of observed fragments are indicated at the right. M, molecular size markers (1,000, 900, 800, 700, 600, 500, 400, 300, and 200 bp, top to bottom, respectively).
FIG. 5
FIG. 5
Splicing of N-ras. In tumor 3, exon −1 is skipped during RNA processing (cryptic splice donor and normal exon 1 splice acceptor sites, located at positions 357 and 1170, respectively; positions correspond to the GenBank sequence with accession no. L19607). In tumors 9 and 11, two mRNAs are generated. Splice product A contains the intron fragment from the provirus to exon 1, and splice product B contains a minor intron 1 fragment (cryptic splice donor and normal exon 1 splice acceptor sites, at positions 1097 and 1170, respectively; positions correspond to the GenBank sequence with accession no. L19607). Provirus-specific primer a (5′-TCCGAATCGTGGTCTCGCTGATCCTTGG-3′) and N-ras primer b (positions 428 to 405; GenBank accession no. X13664) were used in all cases. Arrowheads indicate the locations of the proviruses and proviral sequences. ut, untranslated.
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