Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Oct 10;97(21):11377-82.
doi: 10.1073/pnas.97.21.11377.

A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse

L Conforti  1 , A Tarlton, T G Mack, W Mi, E A Buckmaster, D Wagner, V H Perry, M P Coleman
Affiliations

A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse

L Conforti et al. Proc Natl Acad Sci U S A. .

Abstract

Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, C57BL/Wld(S). Ubiquitin fusion degradation protein 2 (Ufd2) and a previously undescribed gene, D4Cole1e, span the proximal and distal boundaries of the repeat unit, respectively. They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld(S). The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction. Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld(S) brain. This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld. The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland. The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld(S) mice. However, it is undetectable on Northern blots of Wld(S) brain and seems unlikely to be the Wld gene. These data reveal both a candidate gene for Wld and the potential of the Wld(S) mutant for studies of ubiquitin and retinoid metabolism.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Location of exons within the 85-kb WldS triplication repeat unit. In WldS, three adjacent repeat units are shown to indicate (a) how Ufd2 and D4Cole1e are brought together to form a chimeric gene, (b) that normal copies of Ufd2 and D4Cole1e exist still at either end of the repeat array (although the complete genes are not shown), and (c) how Rbp7 is present at three times the normal copy number.
Figure 2
Figure 2
(a) RT-PCR showing the detection in brain of a WldS-specific chimeric transcript between Ufd2 and D4Cole1e. Primers indicated in (d) were used to amplify fragments of 478 bp and 318 bp as shown. Samples derived from different animals are distinguished by the labels WldS 1, WldS 2, etc. (b) RT-PCR showing WldS-specific expression of the chimeric transcript in dorsal root ganglia (DRG) and sciatic nerve. (c) RT-PCR showing the expression of normal transcripts in both 6J and WldS brain for D4Cole1e (Upper) and Ufd2 (Lower) (expected sizes 334 and 710 bp, respectively). Primer sequences: YGK08 = 5′-ACTAGGGCCGTTTGGCTTC-3′; PE36 = 5′-CCTGAACTGGAGCCAGTGTT-3′; PE6 and PE9 as shown in (d). (d) Sequence of the chimeric cDNA and the predicted coding sequence. The vertical arrow indicates the junction between Ufd2-derived sequence and D4Cole1e-derived sequence (Asp-71 is derived from neither protein but formed by the junction). Horizontal arrows indicate the location, and 3′ direction of primers used in (a) and amino acids shown in bold are those used to make peptides for generation of polyclonal antisera 183 (Ile-325 to Lys-339) and 185 (Thr-64 to His-79).
Figure 3
Figure 3
(a) Northern blot showing WldS-specific expression of the chimeric transcript (as detected by a D4Cole1e-specific probe) and a high level of expression in brain. A faint band of 2.3 kb seen in liver in both strains probably corresponds to the normal D4Cole1e transcript. Samples derived from different animals are distinguished by the labels WldS 1, WldS 2, etc. A β-actin control indicates the quantity and integrity of RNA in each lane. (b) Northern blot showing expression of Rbp7 in tissues from C57BL/WldS and C57BL/6J mice. Expression is increased in WldS. (c) Northern blot showing higher expression of Rbp7 in male than in female (nonpregnant) mammary gland and up-regulation during pregnancy. The two right lanes show again the increase during pregnancy.
Figure 4
Figure 4
Western blots of mouse brain homogenate with antisera 183 (a) against a peptide derived from C-terminal region sequence of D4Cole1e and 185 (b) against a peptide whose sequence spans the junction between Ufd2 and D4Cole1e. Each antiserum was used at 1:500 dilution and detects a consistently WldS-specific 43-kDa protein. Antiserum 183 also detects a 40-kDa protein, which may be either nonspecific or the endogenous D4Cole1e product. This additional band indicates similar loading and transfer of in the WldS and C57BL/6J lanes. Samples derived from different animals are distinguished by the labels WldS 1, WldS 2, etc.
Figure 5
Figure 5
Alignment of amino acid sequences of retinoid-binding protein family members and peripheral myelin protein P2, showing the close relationship of Rbp7 with CRBPI and CRBPII. CRABPI, CRABPII, and P2 diverge from the CRBP family particularly at their C-terminal ends. Amino acids that are totally conserved in these proteins are shown in bold, and the percentage identity with Rbp7 is shown at the end. A dash indicates an identical amino acid to Rbp7; periods indicate a gap introduced to optimize the fit.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Lunn E R, Perry V H, Brown M C, Rosen H, Gordon S. Eur J Neurosci. 1989;1:27–33. - PubMed
    1. Waller A. Philos Trans R Soc London. 1850;140:423–429.
    1. Dal Canto M C, Gurney M E. Brain Res. 1995;676:25–40. - PubMed
    1. Fujimura H, Lacroix C, Said G. Brain. 1991;114:1929–1942. - PubMed
    1. Buckmaster E A, Perry V H, Brown M C. Eur J Neurosci. 1995;7:1596–1602. - PubMed

Publication types

MeSH terms