Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults.

We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2 + 3 deletions and t(14;18) translocations as a measure of illegitimate V(D)J recombination. We determined the baseline frequencies of these two mutations in mononuclear leukocyte DNA from the umbilical cord blood of newborns and from the peripheral blood of adults. In an initial group of 21 newborns, no t(14;18) translocations were detected (< 0.049 x 10(-7)). The frequency of HPRT exons 2 + 3 deletions was 0.10 x 10(-7) per mononuclear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%-70%) and previous results using the T-cell cloning assay (approximately 2-3 x 10(-7) per clonable T-cell). Phytohemagglutinin (PHA), as used in the T-cell cloning assay, was examined for its effect on the frequencies of these mutation events in mononuclear leukocytes from an additional 11 newborns and from 12 adults. There was no significant effect of PHA on t(14;18) translocations which were rare among the newborns (1 detected among 2.7 x 10(8) leukocytes analyzed), and which occurred at frequencies from < 1 x 10(-7) (undetected) to 1.6 x 10(-4) among the adults. The extremely high frequencies of t(14;18)-bearing cells in three adults were due mainly to in vivo expansion of two to six clones. However, PHA appeared to stimulate a modest (although not significant) increase in the frequency of HPRT exons 2 + 3 deletions in the leukocytes of the newborns, from 0.07 x 10(-7) to 0.23 x 10(-7). We show that both the direct PCR assay and the T-cell cloning assay detect similar frequencies of HPRT exons 2 + 3 deletions when calculations are normalized to blood volume, indicating that the apparent discrepancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effects of environmental genotoxic agents on this clinically important recombination mechanism.

Targeted recombination at the Chinese hamster APRT locus using insertion versus replacement vectors.

In this study, we have examined the effects of targeting vector configuration and site of vector linearization on the frequency of targeted recombination at the endogenous CHO APRT locus, and have analyzed the types and class distributions of APRT+ recombinants obtained in APRT targeting experiments employing uncut circular, insertion-type (ends-in), and replacement-type (ends-out) configurations of the same pAG7 targeting vector, including configurations produced by introduction of a double-strand break (DSB) at sites either within, or at the 5' or 3' boundaries of APRT targeting homology. Our results suggest that: 1) plasmid-chromosome targeted recombination in mammalian cells may not be stimulated to the same degree by a DSB in the targeting vector as by a DSB in the chromosomal target; 2) recombinant class distributions are highly dependent upon targeting vector configuration; and 3) one-sided invasion mechanisms may play a significant role in homologous recombination in mammalian cells.

Cloning and characterization of 5E6(Ly-49C), a receptor molecule expressed on a subset of murine natural killer cells.

5E6 is a cell surface molecule expressed on a subpopulation of murine natural killer (NK) cells that are involved in the specific rejection of H-2d or H-2f (hemopoietic histocompatibility determinant 2) bone marrow cell grafts. Here, we isolated and cloned the gene encoding 5E6 and determined the nucleotide sequence of the cDNA. 5E6 is nearly identical to Ly-49C; the deduced amino acid sequence reveals a polypeptide of 266 amino acids with a molecular weight of 31,284 that contains multiple cysteine residues to explain its disulfide-linked homodimer structure and five potential N-linked glycosylation sites. 5E6 is a type II integral membrane protein with an extracellular carbohydrate recognition domain characteristic of C-type (Ca(2+)-dependent) animal lectins. Chromosomal mapping indicates that 5E6 is located within the NK gene complex on chromosome 6. The sequence of 5E6 mRNA and the degree of glycosylation of 5E6 protein are under genetic control. Immunoprecipitation before removal of N-linked sugars reveals different size molecules. There are several nucleotide differences among BALB/c, B6, and NZB mRNAs; however, none of them would be expected to affect N-glycosylation. Of particular interest are two findings: (a) BALB/c, B6, and (BALB/c x B6)F1 5E6 reduced molecules are approximately 65, 54, and 54 kD, and (b) the cDNA sequence of (BALB/c x B6)F1 is identical to B6. Thus, there appears to be allelic exclusion of 5E6 expression that may be related to the ability of F1 hybrid mice to reject parental H-2d bone marrow cell grafts.

Correction of chromosomal instability and sensitivity to diverse mutagens by a cloned cDNA of the XRCC3 DNA repair gene.

The mutagen-sensitive CHO line irs1SF was previously isolated on the basis of hypersensitivity to ionizing radiation and was found to be chromosomally unstable as well as cross-sensitive to diverse kinds of DNA-damaging agents. The analysis of somatic cell hybrids formed between irs1SF and human lymphocytes implicated a human gene (defined as XRCC3; x-ray repair cross-complementing), which partially restored mitomycin C resistance to the mutant. A functional cDNA that confers mitomycin C resistance was transferred to irs1SF cells by transforming them with an expression cDNA library and obtaining primary and secondary transformants. Functional cDNA clones were recovered from a cosmid library prepared from a secondary transformant. Transformants also showed partial correction of sensitivity to cisplatin and gamma-rays, efficient correction of chromosomal instability, and substantially improved plating efficiency and growth rate. The XRCC3 cDNA insert is approximately 2.5 kb and detects an approximately 3.0-kb mRNA on Northern blots. The cDNA was mapped by fluorescence in situ hybridization to human chromosome 14q32.3, which was consistent with the chromosome concordance data of two independent hybrid clone panels.

Homology dependence of targeted recombination at the Chinese hamster APRT locus.

Using simple linear fragments of the Chinese hamster adenine phosphoribosyltransferase (APRT) gene as targeting vectors, we have investigated the homology dependence of targeted recombination at the endogenous APRT locus in Chinese hamster ovary (CHO) cells. We have examined the effects of varying either the overall length of targeting sequence homology or the length of 5' or 3' flanking homology on both the frequency of targeted homologous recombination and the types of recombination events that are obtained. We find an exponential (logarithmic) relationship between length of APRT targeting homology and the frequency of targeted recombination at the CHO APRT locus, with the frequency of targeted recombination dependent upon both the overall length of targeting homology and the length of homology flanking each side of the target gene deletion. Although most of the APRT+ recombinants analyzed reflect simple targeted replacement or conversion of the target gene deletion, a significant fraction appear to have arisen by target gene-templated extension and correction of the targeting fragment sequences. APRT fragments with limited targeting homology flanking one side of the target gene deletion yield proportionately fewer target gene conversion events and proportionately more templated extension and vector correction events than do fragments with more substantial flanking homology.

Targeted gene replacement at the endogenous APRT locus in CHO cells.

We demonstrate the feasibility of targeted gene replacement at an endogenous, chromosomal gene locus in cultured mammalian cells, employing a two-step strategy similar to an approach routinely used for genetic manipulation in yeast. Utilizing an APRT+ recombinant generated by targeted integration of plasmid sequences (including a functional copy of the gpt gene) at the CHO APRT locus, we have been able to select gpt- "pop-out" recombinants that have arisen by intrachromosomal recombination between APRT direct repeats at the targeted integration site. Reciprocal exchanges leading to "pop-out" of integrated plasmid/gpt gene sequences occur at a rate of approximately 6.3 x 10(-6) per cell generation. Depending on the site of crossover, such "pop-out" events result in either replacement or restoration of the original APRT target gene sequence.

Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster cells.

We have developed a system that permits analysis of targeted homologous recombination at an endogenous, chromosomal gene locus in cultured mammalian cells. Using a hemizygous, adenine phosphoribosyltransferase (APRT)-deficient, Chinese hamster ovary (CHO) cell mutant as a transfection recipient, we have demonstrated correction of a nonrevertible deletion mutation by targeted homologous recombination. Transfection with a plasmid carrying a fragment of the APRT gene yielded APRT+ recombinants at a frequency of approximately 4.1 x 10(-7). The ratio of targeted recombination to nontargeted integrations of plasmid sequences was approximately 1:4000. Analysis of 31 independent APRT+ recombinants revealed conversions of the endogenous APRT gene, targeted integration at the APRT locus, and a third class of events in which the plasmid donor APRT fragment was converted to a full-length, functional gene.

Endocytosis of lysosomal alpha-galactosidase A by cultured fibroblasts from patients with Fabry disease.

The endocytosis of alpha-galactosidase A was studied in cultured fibroblasts from patients with Fabry disease. Alpha-galactosidase A was purified from human placenta by chromatography on concanavalin A-Sepharose, DEAE-cellulose, and N-epsilon-aminocaproyl-alpha-D-galactosylamine-Sepharose. Separation of the high-uptake form of the enzyme from the low-uptake form was accomplished by chromatography on ECTEOLA-cellulose. With the high-uptake form of the enzyme, the uptake was linear at low concentrations of enzyme and had a Kuptake of 0.01 U/ml of medium that corresponds to a Km of 5.0 x 10(-9) M. At high concentrations of enzyme, it became saturated. The high-uptake form could be converted to the low-uptake form by treatment with acid phosphatase. Mannose-6-P strongly inhibited the active uptake of the enzyme. Once taken up into the lysosomes of Fabry disease fibroblasts, alpha-galactosidase A activity was rapidly lost in the first 2 days of incubation at 37 degrees C, but was fairly stable for the next 6 days. The half-life of internalized alpha-galactosidase A activity was calculated to be 4 days. Crosslinking of the enzyme with hexamethylene diisocyanate did not increase the intracellular stability of alpha-galactosidase A activity.

Differential assay for lysosomal alpha-galactosidases in human tissues and its application to Fabry's disease.

A simple and sensitive fluorometric method has been described for the differential determination of the activity of lysosomal alpha-galactosidase A and alpha-galactosidase B. The procedure employs 4-methylumbelliferyl-alpha-D-galactopyranoside as substrate and N-acetylgalactosamine as an inhibitor of alpha-galactosidase B, but not of alpha-galactosidase A to differentiate the two activities. This method was shown to be applicable in the differentiation of the two enzyme activities in human tissues and in the diagnosis of the heterozygous and hemizygous genotypes for Fabry's disease in cultured skin fibroblasts.