Andre J. Ouellette

Purification and primary structure of murine cryptdin-1, a Paneth cell defensin

We have purified and determined the amino acid sequence of cryptdin‐1, a murine Paneth cell defensin. The peptide corresponds to a previously characterized mRNA that accumulates to high abundance during postnatal ontogeny of the small bowel. Acid‐extracted intestinal protein was fractionated by cation‐exchange chromatography and fractions were assayed for antimicrobial activity. One peak of anti‐Salmonella activity contained a putative defensin, based on its predicted electrophoretic migration in acid‐urea PAGE. The peptide was purified to homogeneity by RP‐HPLC and sequenced. These studies demonstrate defensin expression in non‐myeloid tissue. The N‐terminal extension of cryptdin‐1 is a unique structural feature of this novel epithelial defensin.

Localization of the cryptdin locus on mouse chromosome 8

Cryptdin is a defensin-related peptide, and its mRNA accumulates to high abundance in epithelial cells of intestinal crypts beginning in the second week of postnatal development. The cryptdin (Defcr) locus was assigned to mouse chromosome 8 by Southern blotting of DNAs from mouse/hamster somatic hybrid cell lines. Analysis of somatic hybrid DNAs for mouse-specific restriction fragments showed zero discordance and perfect concordance with chromosome 8. The Defcr locus was localized on chromosome 8 by analysis of DNAs from recombinant inbred (RI) strains of mice after identification of three potential Defcr alleles based on restriction fragment length polymorphisms (RFLPs) in inbred strains. The strain distribution patterns of the Defcr locus were compared with those of chromosome 8 markers in five panels of RI strains. Analysis of cosegregation of Defcr with xenotropic proviral locus Xmv-26 and additional loci confirmed the chromosomal assignment and showed that Defcr is on proximal chromosome 8 within approximately 6 (1.3 to 21.3) cM of Xmv-26. The mouse Defcr locus and the human defensin gene(s) located on chromosome 8p23 appear to map to homologous regions.

Metallothionein mRNA expression in fetal mouse organs

The regulation of metallothionein biosynthesis in mammalian development was investigated by examining organs of 17-day fetal mice for biologically active metallothionein mRNA. Metallothionein was identified in cell-free translation products by migration in polyacrylamide gels and its characteristic elution on Sephadex G-50 columns. Metallothionein constitutes ∼7.5% of [35S]cysteine incorporated into polypeptides directed by mRNA from fetal liver, but it is not detectable in mRNA-directed products of fetal kidney, small bowel, heart, or adult liver. Consistent with a fetal-specific role, hepatic metallothionein mRNA content decreases abruptly in newborn mice, becoming undetectable within 12 days.

Alterations in c-abl gene methylation in cells transformed by phagocyte-generated oxidants

DNA from 10T1/2 cells transformed by activated neutrophils was analyzed for restriction length polymorphisms (RFLPs) in cellular homologues of retroviral oncogenes, and consistent RFLPs were found in MspI sites of the c-abl gene of all PMN-transformed cell lines. MspI digests probed with c-myc, v-Ki-ras, v-Ha-ras or v-mos showed no RFLPs, and none were observed in EcoRI, PstI, HindIII, BamHI, SmaI, Sau3a, MboI, HhaI, or TaqI digests probed with v-abl. Analysis of HpaII digests supports the conclusion that c-abl RFLPs result from differential methylation of the CCGG HpaII/MspI recognition sequence. MspI RFLPs in the c-abl gene may provide markers for oxidant-related genetic injury.

Influence of androgen on translatable renin mRNA in the mouse submandibular gland.

In mature outbred Swiss male mice, submandibular gland renin enzyme activity is 4- and 10-fold higher than in glands of prepubescent males and mature females, respectively. Levels of translatable renin mRNA have been studied in mouse submandibular gland during postnatal development and following administration of testosterone. The [35S]methionine-labeled cell-free translation products directed by male glandular mRNA contain a 47 +/- 2kd renin precursor that is not detected in products coded by prepubescent male or female gland mRNA. This cell-free synthesized precursor is detected immunochemically only in the translation products of gland mRNA from males of 33 days or older and from females receiving testosterone administration, a pattern consistent with the measurements of renin enzyme activity. This increase in biologically active renin mRNA is a selective one, since unfractionated male and female mRNAs have similar overall nucleotide sequence complexity corresponding to 1% of mouse single copy DNA. The cDNA transcribed from male gland mRNA reacts 5- and 10-fold faster with the template mRNA than with female or prepubescent male gland mRNA, respectively, which indicates that the male gland contains abundant nucleotide sequences that exist at low concentration in the female or prepubescent male. Selective hybrid arrested translation confirms that the levels of renin mRNA are lower in the glands of prepubescent males than in those of the mature males. These data indicate that the regulation of renin enzymatic activity by androgens is mediated by an increase in the levels of translatable renin mRNA both during postnatal development and after testosterone administration.

Physical aspects and cytoplasmic distribution of messenger RNA in mouse kidney

As a prerequisite to examining mRNA metabolism in compensatory renal hypertrophy, polyadenylated RNA has been purified from normal mouse kidney polysomal RNA by selection on oligo(dT)-cellulose. Poly(A)-containing RNA dissociated from polysomes by treatment with 10 mM EDTA and sedimented heterogeneously in dodecyl sulfate-containing sucrose density gradients with a mean sedimentation coefficient of 20 S. Poly(A) derived from this RNA migrated at the rate of 6-7 S RNA in dodecyl sulfate-containing 10% polyacrylamide gels. Coelectrophoresis of poly(A) labeled for 90 min with poly(A) labeled for 24 h indicated the long-term labeled poly(A) migrated faster than pulse-labeled material. Twenty percent of the cytoplasmic poly(A)-containing mRNA was not associated with the polysomes, but sedimented in the 40-80 S region (post-polysomal). Messenger RNA from the post-polysomal region had sedimentation properties similar to those of mRNA prepared from polysomes indicating post-polysomal mRNA was not degraded polysomal mRNA. Preliminary labeling experiments indicated a rapid equilibration of radioactivity between the polysomal and post-polysomal mRNA populations, suggesting the post-polysomal mRNA may consist of mRNA in transit to the polysomes.

Ontogeny of class II MHC mRNA in the mouse small intestinal epithelium

MHC Class II (Ia) and invariant chain cooperate in the presentation of exogenous antigen by antigen presenting cells to T-helper cells. Both glycoproteins have been identified in the small intestine of the mature mouse. In this study, we examine the ontogeny of mRNA for three molecules; (Ii31, Ii41 and I-A beta) in whole intestine and in isolated epithelial cells. When RNA from whole intestine was analysed in northern blots using cDNA probe, Ii31 mRNA was present in Day 10 mice and at each 5 day time point thereafter; Ii41 and I-A beta were not detected by this technique. To examine ontogeny of Ii chain mRNA in enterocytes, RNA was purified from an enriched population of epithelial cells isolated after systemic perfusion with 30 mM EDTA in Day 21 and Day 28 and adult mice. Ii chain mRNA was not detected until Day 28 by blot hybridization. Reverse transcription of mRNA and amplification of the resultant cDNA by PCR revealed Ii41 and I-A beta as well as Ii31. RNA from Day 21 epithelial cells required five additional amplification cycles to attain cDNA levels equivalent to those found in Day 28 cells for Ii chain, and 10 additional cycles for I-A beta. In conclusion, Ii31, Ii41 and I-A beta mRNA increase rapidly in the enterocyte after weaning.

The defensin-related murine CRS1C gene: expression in Paneth cells and linkage to Defcr, the cryptdin locus.

The site of defensin-related CRS1C gene expression in mouse small bowel and the chromosomal location of the CRS1C locus, Defcr-rs1, have been determined. CRS1C (cryptdin-related sequence 1C) mRNA is an abundant small intestinal sequence that exhibits extensive similarity to the prepro-coding regions of defensin mRNAs yet does not encode a defensin (A. J. Ouellette and J. C. Lualdi, 1990, J. Biol. Chem. 265: 9831-9837). Using sequence-specific probes, CRS1C mRNA was detected in Paneth cells at the base of intestinal crypts by in situ hybridization. Southern blot analysis of genomic DNAs from inbred and recombinant inbred (RI) mouse strains, also conducted with probes specific for CRS1C, showed that the CRS1C locus maps to the proximal region of Chromosome 8. In 62 RI strains, no discordancies were found between Defcr-rs1 and Defcr, the cryptdin gene. Thus, both the Defcr-rs1 and the Defcr genes are expressed in Paneth cells and both are genetically inseparable within 1.58 cM on Chromosome 8. These studies identify a second defensin-related Paneth cell gene in mice.