SCID in Jack Russell terriers: a new animal model of DNA-PKcs deficiency

We recently described the incidence of a SCID disease in a litter of Jack Russell terriers. In this study, we show that the molecular defect in these animals is faulty V(D)J recombination. Furthermore, we document a complete deficit in DNA-dependent protein kinase activity that can be explained by a marked diminution in the expression of the catalytic subunit DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We conclude that as is the case in C.B-17 SCID mice and in Arabian SCID foals, the defective factor in these SCID puppies is DNA-PKcs. In mice, it has been clearly established that DNA-PKcs deficiency produces an incomplete block in V(D)J recombination, resulting in "leaky" coding joint formation and only a modest defect in signal end ligation. In contrast, DNA-PKcs deficiency in horses profoundly blocks both coding and signal end joining. Here, we show that although DNA-PKcs deficiency in canine lymphocytes results in a block in both coding and signal end joining, the deficit in both is intermediate between that seen in SCID mice and SCID foals. These data demonstrate significant species variation in the absolute necessity for DNA-PKcs during V(D)J recombination. Furthermore, the severity of the V(D)J recombination deficits in these three examples of genetic DNA-PKcs deficiency inversely correlates with the relative DNA-PK enzymatic activity expressed in normal fibroblasts derived from these three species.     

Expression of carbonic anhydrase II (CA II) promoter-reporter fusion genes in multiple tissues of transgenic mice does not replicate normal patterns of expression indicating complexity of CA II regulationin vivo

Although the proximal, 5′ 115 bp of the human carbonic anhydrase II (CA II) gene was sufficient for expression of a reporter gene in some transfected cell lines, we found previously that 1100 bp of this promoter (or 500 bp of the mouse CA II promoter) was not sufficient for expression in transgenic mice. We have now studied the expression of linked reporter genes in mice transgenic for either (1) 11 kb of the human 5′ promoter or (2) 8 kb of the human 5′ promoter with mouse sequences from the first exon, part of the first intron (since a CpG island spans this region), and the 3′ sequences of the gene. Expression was found in both cases, but the tissue specificity was not appropriate for CA II. Although there was a difference in the sensitivity of the assays used, the first construct led to expression in many tissues, while the second construct was expressed only in spleen. These findings indicate considerable complexity of DNA control regions for in vivo CA II expression.     

The nucleotide sequence and derived amino acid sequence of cDNA coding for mouse carbonic anhydrase II

The nucleotide sequence of a clone containing mouse carbonic anhydrase (CA) cDNA in pBR322 has been determined. The cloned cDNA contains all of the coding region except for nucleotides specifying the first eight amino acids, and all of the 3' noncoding region, which consists of 700 nucleotides. A cDNA clone was identified which contains an additional 54 bp at the 5' end, so that the complete amino acid sequence of mouse CA could be deduced. This sequence showed a 73-81% homology with other mammalian CA form II isozymes, 56-63% with form I isozymes, and 52-56% with form III isozymes. By examination of the amino acids which are unique and invariant for each isozyme, the mouse amino acid sequence was found to contain 16 of the 23 residues that are unique and invariant to mammalian CA form II isozymes, but only one or no residue for forms I and III, respectively.     

Gene-specific universal mammalian sequence-tagged sites: Application to the canine genome

We are developing a genetic map of the dog based partly upon markers contained within known genes. In order to facilitate the development of these markers, we have used polymerase chain reaction (PCR) primers designed to conserved regions of genes that have been sequenced in at least two species. We have refined the method for designing primers to maximize the number that produce successful amplifications across as many mammalian species as possible. We report the development of primer sets for 11 loci in detail:CFTR, COL10A1, CSFIR, CYP1A1, DCN1, FES, GHR, GLB1, PKLR, PVALB, andRB1. We also report an additional 75 primer sets in the appendices. The PCR products were sequenced to show that the primers amplify the expected canine genes. These primer sets thus define a class of gene-specific sequence-tagged sites (STSs). There are a number of uses for these STSs, including the rapid development of various linkage tools and the rapid testing of genomic and cDNA libraries for the presence of their corresponding genes. Six of the eleven gene targets reported in detail have been proposed to serve as “anchored reference loci” for the development of mammalian genetic maps [O'Brien, S. J.,et al., Nat. Genet. 3:103, 1993]. The primer sets should cover a significant portion of the canine genome for the development of a linkage map. In order to determine how useful these primer sets would be for the other genome projects, we tested the 11 primer sets on the DNA from species representing five mammalian orders. Eighty-four percent of the gene-species combinations amplified successfully. We have named these primer sets “universal mammalian sequence-tagged sites” because they should be useful for many mammalian genome projects.     

Carbonic anhydrase II deficiency: single-base deletion in exon 7 is the predominant mutation in Caribbean Hispanic patients.

To date, three different structural gene mutations have been identified in patients with carbonic anhydrase II deficiency (osteopetrosis with renal tubular acidosis and cerebral calcification). These include a missense mutation (H107Y) in two families, a splice junction mutation in intron 5 in one of these families, and a splice junction mutation in intron 2 for which many Arabic patients are homozygous. We report here a novel mutation for which carbonic anhydrase II-deficient patients from seven unrelated Hispanic families were found to be homozygous. The proband was a 2 1/2-year-old Hispanic girl of Puerto Rican ancestry who was unique clinically, in that she had no evidence of renal tubular acidosis, even though she did have osteopetrosis, developmental delay, and cerebral calcification. She proved to be homozygous for a single-base deletion in the coding region of exon 7 that produces a frameshift that changes the next 12 amino acids before leading to chain termination and that also introduces a new MaeIII restriction site. The 27-kD truncated enzyme produced when the mutant cDNA was expressed in COS cells was enzymatically inactive, present mainly in insoluble aggregates, and detectable immunologically at only 5% the level of the 29-kD normal carbonic anhydrase II expressed from the wild-type cDNA. Metabolic labeling revealed that this 27-kD mutant protein has an accelerated rate of degradation. Six subsequent Hispanic patients of Caribbean ancestry, all of whom had osteopetrosis and renal tubular acidosis but who varied widely in clinical severity, were found to be homozygous for the same mutation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ectopic expression of chloramphenicol acetyltransferase (CAT) in the cerebellum in mice transgenic for a carbonic anhydrase II promoter-CAT construct that is without apparent phenotypic effect

We have developed six transgenic lines of mice with constructs containing presumptive 5' regulatory regions of carbonic anhydrase II (CA II). Four of the lines contained 1,100 bases of the 5' flanking region of the human CA II gene, and two transgenic lines resulted from a construct containing 500 bases of the 5' flanking region of the mouse CA II gene. Tissue-specific expression of the chloramphenicol acetyltransferase (CAT) gene was not obtained in any of the transgenic lines. One of the transgenic lines was found to have high levels of expression of CAT in cerebellum. This expression persisted through multiple generations and was independent of the parental origin of the transgene. On the assumption that the expression was due to the insertion of the transgene in or near a gene expressed normally in cerebellum, homozygous mice were bred for the transgenic insert to see if a mutation might have been induced. Homozygous mice were found and seemed to be normal in all aspects of their phenotype studied. Thus, in this case, neither the insertion of the gene nor the ectopic expression of CAT seemed to be harmful to the animals.     

Dynamics of Cdk1 substrate specificity during the cell cycle

Cdk specificity is determined by the intrinsic selectivity of the active site and by substrate docking sites on the cyclin subunit. There is a long-standing debate about the relative importance of these factors in the timing of Cdk1 substrate phosphorylation. We analyzed major budding yeast cyclins (the G1/S-cyclin Cln2, S-cyclin Clb5, G2/M-cyclin Clb3, and M-cyclin Clb2) and found that the activity of Cdk1 toward the consensus motif increased gradually in the sequence Cln2-Clb5-Clb3-Clb2, in parallel with cell cycle progression. Further, we identified a docking element that compensates for the weak intrinsic specificity of Cln2 toward G1-specific targets. In addition, Cln2-Cdk1 showed distinct consensus site specificity, suggesting that cyclins do not merely activate Cdk1 but also modulate its active-site specificity. Finally, we identified several Cln2-, Clb3-, and Clb2-specific Cdk1 targets. We propose that robust timing and ordering of cell cycle events depend on gradual changes in the substrate specificity of Cdk1.