Elizabeth B. Keller

Protein chain initiation by methionyl-tRNA.

Abstract Wheat germ contains three chromatographically distinct methionine tRNAs, of which two species, one major and one minor, appear to function in protein chain initiation as demonstrated by the AUG-dependent reaction with puromycin on wheat germ ribosomes. Wheat germ extracts contain a very active transformylase which formylates the minor methionyl-tRNA. The major initiating methionyl-tRNA is not formylated or otherwise modified by wheat germ extracts. We postulate that protein chain initiation in the cytoplasm of wheat germ can be brought about by the major initiating methionyl-tRNA without prior formylation or other modification of the α amino group.

Two distinct poly(A) polymerases in yeast nuclei

Abstract Yeast extracts have a poly(A) polymerase activity which catalyzes the addition of adenylate residues to the 3′-terminus of primer RNA. On purification of this activity, two poly(A) polymerases were found. Poly(A) polymerase I elutes from a DEAE-cellulose column at very low salt concentration and prefers as primer an RNA without an oligo(A) segment at the 3′-terminus. Poly(A) polymerase II elutes at a higher salt concentration from DEAE-cellulose and has a requirement for a primer with an oligo(A) segment at the 3′-terminus. The two enzymes are localized largely in the nucleus. It is suggested that these two enzymes function sequentially, first I then II, in forming the long poly(A) sequences that are found on messenger RNAs in yeast.

High efficiency transfection of monkey kidney COS-1 cells

Methods are described for achieving high efficiency transient transfection of COS-1 cells. A transfection solution of vector DNA and DEAE-dextran in phosphate buffered saline is added to the cells, followed by treatment with culture medium containing chloroquine, resulting in a maximum efficiency of about 50%. The high efficiency obtained is primarily dependent on the use of Ca2+- or Mg2+-free buffer solutions for transfection, because the addition of these ions greatly reduces efficiency. Shocking the cells with dimethylsulfoxide does not increase transfection efficiency. COS-1 cells are monkey kidney cells that have a genomic insertion of the SV40 T antigen gene, allowing plasmid expression vectors bearing an SV40 replication origin to be amplified in these cells. Therefore this transfection method is useful for optimizing transient expression of genes in a mammalian cell system.

The TATA-dependent and TATA-independent promoters of the Drosophila melanogaster actin 5C-encoding gene

The major cytoskeletal actin of Drosophila melanogaster, actin 5C, is encoded by a gene (act5C) that has two promoters which are differentially controlled and possess distinct sets of regulatory elements. The distal basal promoter has a TATA motif, but the proximal does not. The distal strong positive domain, centered at nucleotide -290, can be shifted and fused directly to the distal basal promoter without losing its activity. It can also activate heterologous basal promoters containing either TATAAAT or TATTTAA signal when directly fused to them, but cannot activate the basal proximal promoter, which is TATA-less. When the entire distal regulatory region, which includes a remote enhancer-like region, is fused to the proximal promoter, it does not increase the proximal promoter activity. Fusion of the distal strong negative domain to the proximal promoter does not inhibit activity. Thus, all the three major strong regulatory domains of the distal promoter are unable to act on the proximal promoter.

Unique nucleotide sequence adjacent to the poly(A) region in yeast RNA

Abstract Yeast cells growing in a low phosphate medium were labeled with a pulse of 32Pi or [3H]adenine and harvested after 15 minutes. Total RNA was extracted and digested with ribonuclease T1. Poly(A)-rich fragments were isolated from the digest by hybridization to poly(U) impregnated fiberglass filters. Gel filtration showed the fragments to have a uniform chain length of about sixteen. Analysis of the composition gave (A11, C4, U). Complete pancreatic ribonuclease and partial spleen phosphodiesterase digests gave the sequence of the 5′ end of the fragment as CpApApUp-. Since the fragment was a ribonuclease T1 product, the data points to a unique sequence of at least five residues, -GpCpApApUp-, adjacent to the poly(A)-rich terminus of pulse-labeled yeast mRNA. The remainder of the poly(A)-rich fragment consists of A residues with a few randomly interspaced C residues. The known specificity of yeast poly(A) polymerase can account for the presence of C residues in poly(A) tracts.