The mCAT-1 gene encodes a basic amino acid transporter that also acts as the receptor for murine ecotropic leukemia viruses. Targeted mutagenesis in embryonic stem cells has been used to introduce a germ-line null mutation into this gene. This mutation removes a domain critical for virus binding and inactivates amino acid transport activity. Homozygous mutant pups generated from these cells were -25% smaller than normal littermates, very anemic, and died on the day of birth. Peripheral blood from homozygotes contained 50% fewer red blood cells, reduced hemoglobin levels, and showed a pronounced normoblastosis. Histological analyses of bone marrow, spleen, and liver showed a decrease in both erythroid progenitors and mature red blood cells. Mutant fetal liver cells behaved normally in in vitro hematopoietic colony-forming assays but generated an anemia when transplanted into irradiated C.B.-17 SCID mice. Furthermore, reconstitution of the white cell compartment of SCID mice by mutant fetal liver cells was less complete than that observed with a mixed population of wild-type and heterozygous fetal liver cells. Primary embryo fibroblasts from mutant mice were completely resistant to ecotropic retrovirus infection. Thus, mCAT-1 not only appears to be the sole receptor for a group of murine ecotropic retroviruses associated with hematological disease but also plays a critical role in both hematopoiesis and growth control during mouse development.

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A number of bifunctional chemical mutagens induce exocyclic DNA lesions. For example, 2-chloroacetaldehyde (CAA), a metabolite of vinyl chloride, readily reacts with single-stranded DNA to predominantly form etheno lesions. Here, we report on in vivo mutagenesis caused by CAA treatment of DNA in vitro. These experiments used partially duplex phage M13AB28 replicative form DNA in which a part of the lacZ gene sequence was held in single-stranded form to direct reaction with CAA. CAA-treated partial duplex DNA was transfected into Escherichia coli, and the induced base changes were defined by DNA sequencing. These experiments suggested that CAA treatment induced mutations at cytosines, much less efficiently at adenines, but not at guanines or thymines. Among mutations targeted to cytosine, 80% were C-to-T transitions and 20% were C-to-A transversions. Application of a post-labeling method detected dose-dependent formation of ethenoadenine and ethenocytosine in CAA treated DNA. These data indicate that ethenocytosine is a highly efficient mutagen with properties suggestive of a non-instructional DNA lesion in vivo. Paradoxically, ethenoadenines are efficiently bypassed by a mechanism which appears to be largely nonmutagenic.

In order to examine the mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position, the replicative form DNA of phage M13AB28 (mp8 without the amber codons in phage genes) was modified in vitro with aflatoxin B1-2,3-dichloride and transfected into appropriate Escherichia coli cells. Forward mutations in the lacZ alpha-complementing gene segment were identified as light blue or colorless plaques on appropriate indicator plates, isolated, and defined by DNA sequencing. Transfection of modified DNA into uvrA-/mucAB+ cells without prior UV (SOS) induction increased mutation frequency eight-fold over untreated DNA, whereas this increase was 12-fold upon SOS induction. Transfection of modified DNA after conversion of the primary guanine-aflatoxin lesions to the stable imidazole ring-opened formamidopyrimidine-aflatoxin suggested that these lesions were nearly equally mutagenic. A majority of point mutations under all conditions affected G:C bp. Base substitutions were in the majority, but significant frameshift mutagenesis was also detected in SOS-induced cells. Both G-to-T transversions and G-to-A transitions were produced at equal efficiency and together accounted for virtually all of the base substitutions induced by the primary lesions. Point mutations occurred predominantly at predicted damage hotspots. The characteristics of base substitution and frameshift mutations, together with available information point to multiple mechanisms of mutagenesis by this class of mutagens. The data indicate that primary lesions have the properties of both a noninstructional and pseudo-instructional lesion. In addition, the sequence context appears to play a role in determining whether a frameshift or a base substitution is induced by this bulky lesion.