The reduced expression of 6Ckine in the plt mouse results from the deletion of one of two 6Ckine genes - PubMed (original) (raw)

The reduced expression of 6Ckine in the plt mouse results from the deletion of one of two 6Ckine genes

G Vassileva et al. J Exp Med. 1999.

Abstract

6Ckine is an unusual chemokine capable of attracting naive T lymphocytes in vitro. It has been recently reported that lack of 6Ckine expression in lymphoid organs is a prominent characteristic of mice homozygous for the paucity of lymph node T cell (plt) mutation. These mice show reduced numbers of T cells in lymph nodes, Peyer's patches, and the white pulp of the spleen. The genetic reason for the lack of 6Ckine expression in the plt mouse, however, has remained unknown. Here we demonstrate that mouse 6Ckine is encoded by two genes, one of which is expressed in lymphoid organs and is deleted in plt mice. A second 6Ckine gene is intact and expressed in the plt mouse.

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Figures

Figure 1

Figure 1

PCR and Southern blot analysis of the 6Ckine gene locus reveals the existence of two 6Ckine genes, only one of which is detected in the plt mouse. (A) EcoRI-digested DNA from 6CKBAC1, 6CKBAC2, plt, and 129/sv mice was analyzed by Southern blot analysis using a 32P-labeled NheI/BstXI fragment (271 bp) as a probe (probe B, Fig. 3 C). The probe specifically hybridized to 6- and 1.35-kb fragments in 6CKBAC1 DNA, 3- and 1.2-kb fragments in 6CKBAC2, 6- and 1.35-kb fragments in plt mouse genomic DNA, and all four fragments in 129/sv genomic DNA. (B) PCR analysis: PCR primers GV104/GV125 (see Fig. 2) were used to amplify a segment of the 3′ region of the 6Ckine gene. The resulting PCR products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide. Two bands of 1.35 and 1.2 kb were detected in a PCR reaction from genomic DNA from several mouse strains (as indicated). Single bands of 1.35 or 1.2 kb were detected from 6CKBAC1 and 6CKBAC2 DNA, respectively, whereas a single band of 1.35 kb was amplified from the plt mouse genomic DNA.

Figure 2

Figure 2

(A) The 6Ckine genes found on the two BACs are distinct. A schematic of the sequence of SacI fragments (7.5 kb) from 6CKBAC1 (leu) and 6CKBAC2 (ser) is shown. Black and white boxes represent 6Ckine exons. Open triangles indicate larger deletions. Single base differences outside of the exons are not shown. The four nucleotide differences identified in coding regions (black boxes) and noncoding regions (white box) of the two 6Ckine genes are shown. Vertical arrows identify the amino acid difference at position 65. Horizontal arrows indicate the positions of the PCR primers used for analysis of 6Ckine gene locus. nt, nucleotide. (B) Sequences of the intron–exon boundaries are identical between the two copies with the exception of the intron2–exon3 junction. The 6Ckine-ser sequence is added to the intron2–exon3 boundary sequence to illustrate the single nucleotide difference (arrow). These sequence data are available from EMBL/GenBank/DDBJ under accession numbers AF171085 and AF171086 for 6Ckine-leu and 6Ckine-ser, respectively.

Figure 3

Figure 3

Southern blot analysis of 6Ckine genomic loci suggests that the 6Ckine-ser gene of the plt mouse contains significant deletions. (A) EcoRI-digested genomic DNA from 6CKBAC1 and 6CKBAC2 and genomic DNA from 129/sv and plt mice were probed with a 0.8-kb SacI/NheI fragment (probe A, indicated). An 8-kb fragment was recognized in 6CKBAC1, and a 6.5-kb fragment was recognized in 6CKBAC2. The plt mouse genomic DNA contained only an 8-kb fragment DNA, whereas both 8- and 6.5-kb fragments hybridized in 129/sv DNA. (B) HindIII-digested DNA from 6CKBAC1 and 6CKBAC2 and genomic DNA from 129/sv and plt mice was probed with a 271-bp NheI/BstXI fragment (probe B, indicated). A 9-kb fragment hybridized in 6CKBAC1 and an 11-kb fragment hybridized in 6CKBAC2 DNA. Only a 9-kb fragment was recognized in plt genomic DNA, whereas both 9- and 11-kb fragments were present in 129/sv DNA. (C) Schematic of the 6Ckine genomic locus, with positions of initiation and stop codons indicated by ○ and •, respectively. The positions of the hybridization probes are indicated by shaded boxes, and the sizes of the fragments recognized by these probes relative to each BAC are indicated.

Figure 4

Figure 4

6Ckine is expressed in both wild-type and plt mice. Total RNA (20 μg/lane) from either BALB/c (A) or plt (C) mouse tissues was hybridized with a 32P-labeled 6Ckine probe and subjected to autoradiography. BALB/c mice showed 6Ckine expression in various lymphoid and nonlymphoid tissues, whereas plt mice showed expression only in nonlymphoid tissue. Total spleen RNA (20 μg) from BALB/c mice was included as a positive control in each blot. Ethidium bromide–stained gel of BALB/c and plt RNA is shown in B and D, respectively.

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