A second polymorphic lens crystallin (LEN-2) in the mouse: genetic and biochemical analysis of LEN-1 and LEN-2.

Two electrophoretic polymorphisms affecting lens crystallins, designated LEN-1 and LEN-2, have been discovered among inbred strains of mice. Analysis by isoelectric focusing demonstrated that both crystallins are monomeric proteins with isoelectric points at or above pH 7. Both proteins eluted in the low molecular weight (LM) fraction upon Sephadex G-200 gel filtration but LEN-2 was shown to be larger than LEN-1 by G75SF gel filtration and denaturing gel electrophoresis. Linkage analysis demonstrated that the genes encoding LEN-1 and LEN-2 assort independently. Amino acid analysis of the allelic products of the two genes revealed that genetic variants of each respective crystallin were very similar in amino acid compositions but that LEN-1 and LEN-2 were dissimilar crystallins.

Analysis of a mouse alpha-globin gene mutation induced by ethylnitrosourea.

A DBA/2 mouse treated with ethylnitrosourea sired an offspring whose hemoglobin showed an extra band following starch gel electrophoresis. The variant hemoglobin migrated to a more cathodal position in starch gel. Isoelectric focusing indicated that chain 5 of the mutant hemoglobin migrated to a more cathodal position than the normal chain 5 from DBA/2 mice and that the other alpha-globin, chain 1, was not affected. On focusing gels the phenotype of the mutant allele, Hbay9, was expressed without dominance to normal chain 5, and Hbay9/Hbay9 homozygotes were fully viable in the laboratory. The molecular basis for the germinal mutation was investigated by analyzing the amino acid sequence of chain 5y9, the mutant form of alpha-chain 5. A single amino acid substitution (His leads to Leu) at position 89 was found in chain 5y9. We propose that ethylnitrosourea induced an A leads to T transversion in the histidine codon at position 89 (CAC leads to CTC). This mutation has apparently not been observed previously in humans, mice or other mammals, and its novel occurrence may be indicative of other unusual mutational events that do not ordinarily occur in the absence of specific mutagen exposure.

The primary structure of genetic variants of mouse hemoglobin.

The primary structures of the alpha globins from CE/J, DBA/2J, and a stock of Potter's mice were determined to identify the amino acid substitutions associated with the unique isoelectric focusing patterns of these hemoglobins. In addition, the primary structures of the alpha globins from MOL III and PERU mice were studied in search of amino acid substitutions that may not be detected by isoelectric focusing. CE/J hemoglobin contains a unique kind of alpha globin called chain 5. It differs from the single kind of alpha globin (chain 1) in C57BL/6 by having alanine rather than glycine at position 78. DBA/2J hemoglobin has two kinds of alpha globins: one half is like chain 5 and the other half is like chain 1. The hemoglobin from Potter's stock of Mus musculus molossinus also contains chains 1 and 5, but they are expressed at different levels i.e., 80% chain 1 and 20% chain 5. MOL III hemoglobin has a single kind of a alpha globin identical to that in C57BL/6, and PERU hemoglobin contains approximately 40% chain 1 and 60% chain 4. Chains 1 and 4 have different amino acids at positions 25, 62 and 68. These studies confirm that mouse hemoglobins separable by isoelectric focusing, but not by other means of electrophoresis, have substitutions of neutrally charged amino acids in their alpha chains.

Differential association of transfer RNAs with the genomes of murine, feline and primate retroviruses.

The tRNAs that are bound to the genomic RNAs of several murine, feline, and primate retroviruses have been identified. Transfer RNAs were divided into those loosely bound and those tightly bound by stepwise thermal dissociation of the 70 S RNA. They were then identified and semiquantitated by aminoacylation. Proline tRNA is the most tenaciously bound tRNA in several strains of murine leukemia virus, two strains of feline leukemia virus, and the primate viruses simian sarcoma, baboon endogenous, and gibbon ape lymphoma. In the feline xenotropic virus, RD-114, tRNAGly is enriched in the most tightly bound fraction. In Mason-Pfizer monkey virus, as in the murine mammary tumor virus, tRNALys is the tRNA most tenaciously bound to its genomic RNA. Besides the most tightly associated tRNA, one or more different tRNAs are found in relatively large amounts in association with the 70 S RNA. (For convenience, we refer to the largest RNA ccomplex (50-70 S) isolated from any of the retroviruses studies as '70 S' RNA.) These tRNAs can be distinguished from the most tightly bound tRNA by the fact that they can be dissociated at lower temperatures. However, they occur in the same relative abundance as the tightly bound tRNA.

Species comparison of protein synthesis accuracy.

The average accuracy of protein synthesis in reticulocytes from several mammalian species does not correlate with longevity potential from 13 to 90 years. Isoleucine incorporation into highly purified hemoglobin chains which contain no genetically coded isoleucine was used as a direct test of protein synthesis accuracy. Since isoleucine can be incorporated into these molecules by mutations in a few cells as well as errors in most cells, the constant level of isoleucine substitution may also show that the mutation rates are not dramatically different among these species. Isoleucine substitutions in hemoglobin can be used to estimate mutations only above the level of errors, which may be as low as 1/1,000,000, but the probability of seeing a few mutant clones at any time is dependent on the number of stem cells producing reticulocytes. The number of stem cells being expressed is a reflection of the number of cell divisions per clone. If the number of cell divisions per clone is 30 or less, then isoleucine substitutions would increase when the mutation accumulation rose above 30 per million for the mutation to isoleucine at any position in the alpha or beta chain.

tRNA's associated with the 70S RNA of avian myeloblastosis virus.

The distribtuion of various amino acid tRNA's in the 4S RNA components of avian myeloblastosis virus (AMV) and in 4S RNA prepared from chicken cmbryo cells, chicken myeloblasts, and chicken livers was determined. This was done by aminoacylating the 4S RNA samples with a mixture of 17 radioactive amino acids and subsequently identifying the tRNA-accepted amino acids on an amino acid analyzer after deacylation. In embryo cells, myeloblasts, and liver, tRNA's accepting all 1m amino acids were demonstrated. "Free" AMV 4S RNA was characterized by very low quantities of glutamate, valine, and tyrosine tRNA's. RNAs accepting all 17 amino acids, with the exception of tyrosine, were shown to be present in the "70S-associated" 4S RNA which dissociates at 60 C. The bulk of the 70S-associated 4S RNA was dissociated at 60 C at low ionic strength with a concomitant conversion of 70S RNA to 35S RNA. The residual associated 4S RNA was dissociated by further heating of the 35S RNA to 80 C; tryptophan tRNA accounted for greater than 90% of the total amino acid accepting activity in this fraction. The results support other studies in suggesting that tryptophan tRNA may serve as a primer for DNA synthesis in AMV, as has been shown in Rous sarcoma virus.