Emanuel Goldman

Low-usage codons in Escherichia coli, yeast, fruit fly and primates

Codon usage is compared between four classes of species, with an emphasis on characterization of low-usage codons. The classes of species analyzed include the bacterium Escherichia coli (ECO), the yeast Saccharomyces cerevisiae (YSC), the fruit fly Drosophila melanogaster (DRO), and several species of primates (PRI) (taken as a group; includes eleven species for which nucleotide sequence data have been reported to GenBank, however, greater than 90% of the sequences were from Homo sapiens). The number of protein-coding sequences analyzed were 968 for ECO, 484 for YSC, 244 for DRO, and 1518 for PRI. Three methods have been used to determine low-usage codons in these species. The first and most common way of assessing codon usage is by summing the number of time codons appear in reading frames of the genome in question. The second way is to examine the distribution of usage in different genes by scoring the number of protein reading frames in which a particular codon does not appear. The third way starts with a similar notion, but instead considers combinations of codons that are missing from the maximum number of genes. These three methods give very similar results. Each species has a unique combination of eight least-used codons, but all species contain the arginine codons, CGA and CGG. The agreement between YSC and PRI is particularly striking as they share six low-usage codons. All six carry the dinucleotide sequence, CG. The eight least-used codons in PRI include all codons that contain the CG dinucleotide sequence. Low-usage codons are clearly avoided in genes encoding abundant proteins for ECO, YSC DRO. In all species, proteins containing a high percentage of low-usage codons could be characterized as cases where an excess of the protein could be detrimental. Low codon usage is relatively insensitive to gross base composition. However, dinucleotide usage can sometimes influence codon usage. This is particularly notable in the case of CG dinucleotides in PRI.

Synthesis of homocysteine thiolactone by methionyl-tRNA synthetase in cultured mammalian cells

Homoeysteine thiolactone is a product of an error‐editing reaction, catalyzed by Escherichia coli and Saccharomyces cerevisiae methionyl‐tRNA synthetases, which prevents incorporation of homocysteine into tRNA and protein both in vitro and in vivo. Here, homocysteine thiolactone is also shown to be synthesized by cultured mammalian cells such as human cervical carcinoma (HeLa), mouse renal adenocarcinoma (RAG), and Chinese hamster ovary (CHO) cells labeled with [35S]methionine, but not by normal human and mouse (Balb/c 3T3) fibroblasts. A temperature‐sensitive methionyl‐tRNA synthetase mutant of CHO cells, Met‐1, does not make the thiolactone at the non‐permissive temperature. The data indicate that methionyl‐tRNA synthetase is involved in synthesis of homocysteine thiolactone in CHO cells, thereby extending this important proofreading mechanism to mammalian cells.

Analysis of host range of nontransforming polyoma virus mutants.

Abstract The nontransforming polyoma virus mutant NG-18, screened originally for its ability to grow in polyoma-transformed cells, has now been shown to grow in a variety of other cell types. Among cells found to be substantially permissive for the growth of NG-18 are: Phenotypic revertants of polyoma-transformed 3T3 cells, C-type RNA virus transformed and/or producing 3T3 cells, 3T3 cells that become transformed upon infection with leukemia virus, primary baby mouse kidney epithelial cells, and primary mouse embryo fibroblasts. Among cells found to be poor hosts for the growth of NG-18 are: Several 3T3 cell lines, SV40-transformed 3T3 cells, spontaneously transformed 3T3 cells, radiation- and chemical carcinogen-transformed 3T3 cells, and late passage mouse embryo fibroblasts. Similar results have been obtained using three other independently isolated host range mutants of the same type as NG-18. The permissive state thus does not require a cell-associated polyoma genome as the basis for complementation of growth for this class of host range mutant. In addition, the results demonstrate that cells can be permissive for the growth of such nontransforming virus mutants without themselves possessing properties commonly associated with transformation: Loss of density-dependent inhibition of growth, low serum requirement for growth, loss of anchorage dependence of growth, and lectin agglutinability. Implications of these findings are discussed in reference to possible mechanisms of action of the NG-18 gene in productive infection and transformation.

Uncharged tRNA, protein synthesis, and the bacterial stringent response.

Uncharged tRNA has been shown in vivo to have an active role both in the stringent response, and in modulating the rate of translational elongation. Both of these effects appear to be mediated by codon–anticodon interactions on the ribosome. Although the involvement of uncharged tRNA in the stringent response was expected from in vitro experiments, it has only recently been confirmed in vivo. Inhibition of translation by cognate uncharged tRNA was not expected, and a model is proposed in which excess uncharged tRNA competes with charged tRNA (in ternary complex) for the 30S component of the ribosomal A site. When uncharged tRNA is in sufficient excess over charged tRNA, interaction of uncharged tRNA with the 50S component of the A site occurs as well, leading to a stringent response. The cell has a continuum of responses to decreasing aminoacyl‐tRNA levels: in moderately limited conditions, the proportion of uncharged tRNA increases, and the translation rate is slowed; under more severe limitations, uncharged tRNA provokes a stringent response, with pleiotropic consequences for the cell.

Specificity of codon recognition by Escherichia coli tRNALeu isoaccepting species determined by protein synthesis in vitro directed by phage RNA

Codon-anticodon recognition and transfer RNA utilization for the leucine tRNA isoaccepting species of Escherichia coli have been studied by protein synthesis in vitro directed by sequenced bacteriophage MS2 RNA. We have added radioactive Leu-tRNALeu isoaccepting species as tracers, rather than use a tRNA-dependent system, since in the presence of an excess of non-radioactive leucine, there is no transfer of radioactive leucine from one isoaccepting species to another. MS2-specific peptides containing leucine residues encoded by known codons were isolated and identified, and the relative abilities of the Leu-tRNALeu isoaccepting species to transfer leucine into these peptides compared. Sequenced tRNA1Leu and sequenced tRNA3Leu are of roughly equal efficiency in their ability to recognize CUC and CUA codons, while tRNA3Leu is highly preferred for the CUU codon; tRNA4Leu and tRNA5Leu both recognize UUA and UUG codons, with tRNA4Leu slightly preferred for the UUA codon. We conclude that: (1) wobble is greater than permitted by the wobble hypothesis; (2) there is still some discrimination in the third code letter, and that the CUX4 (CUC, CUA, CUU, CUG) portion of the leucine family of six codons is not read by a simple “two out of three” mechanism; (3) a Watson-Crick pair (C · G) between codon and anticodon does not appear to be preferred over an unorthodox pair (C · C) in the wobble position; (4) a standard wobble pair (U · G) between codon and anticodon is preferred over an unorthodox pair (U · C); and (5) the extensive wobble observed in the CUX4 leucine codon series is not paralleled in the UUX4 leucine (UUG, UUA) and phenylalanine (UUU, UUC) codon series, where mistranslation would be the consequence of such wobble.

Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display

An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or –1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a –1 reading frame‐shift, and one open reading frame) in gene fusions to a reporter β‐galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non‐open reading frame constructs gave (surprisingly) high expression of the reporter (10–40% of control vector expression levels) in two out of the three frames. A site‐directed mutant of the TGA stop codon (to TTA) in the + 1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site‐directed mutant of the TGA in the –1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site‐directed mutant of the + 1 shifter, primarily expressed the reporter only in the zero frame.—Goldman, E., Korus, M., Mandecki, W. Efficiencies of translation in three reading frames of unusual non‐ORF sequences isolated from phage display. FASEB J. 14, 603–611 (2000)

Lack of correlation between agglutinability, the surface distribution of con A and post-confluence inhibition of cell division in ten cell lines

Agglutinability by concanavalin A, distribution of surface-bound concanavalin A, and maximal cell density in monolayer culture were examined under similar conditions in parallel cultures of ten established cell lines. The degree of agglutinability of the cell lines did not correlate with the presence or absence of patching of concanavalin A bound to the cell surface, as determined with a hemocyanin marker. Agglutinability was also not always correlated with the loss of post-confluence inhibition of cell division. Two clones of mouse 3T3 fibroblasts that maintained post-confluence inhibition of cell division and low agglutinability differed substantially with respect to the surface distribution of concanavalin A. Patching of concanavalin A binding sites is neither necessary nor sufficient to explain differences in agglutinability between cell lines.

Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.

Specificity of protein synthesis by bacterial ribosomes and initiation factors: Absence of change after phage T4 infection☆

Abstract Using several natural messenger RNAs—f2 RNA, Qβ RNA, T7 RNA, T4 early mRNA, T4 late mRNA and Escherichia coli RNA—ribosomes isolated from cells either 5 or 12 minutes after T4 infection direct synthesis of only 35 to 70% as much protein as do ribosomes from uninfected cells. However, with poly(U) or formaldehyde-treated f2 RNA message, both types of ribosomes work equally well. Experiments mixing salt-washed ribosomes and initiation factors from these cells show, in agreement with work of others, that the reduction with natural messages is due only to changes in the initiation factors. As shown by peptide mapping of protein made in vitro and labeled with N -formyl [ 35 S]methionyl-transfer RNA, T4 and control ribosomes direct initiation, using f2 RNA, Qβ RNA, early and late T4 mRNA and T7 RNA, of the same polypeptides, in about the same relative proportions. We conclude that there is no change in the specificity of initiation factors after T4 infection; alterations in the amounts of factors are probably not essential either for shut-off of host protein synthesis or for the transition from early to late T4 protein synthesis.

Antibiotic Abuse in Animal Agriculture: Exacerbating Drug Resistance in Human Pathogens

ABSTRACT Most of the antibiotics produced in the U.S. are fed to farm animals routinely as “growth promoters,” and to facilitate “factory farming.” Unfortunately, this places selective pressure on bacteria to develop antibiotic-resistance. Genes that neutralize antibiotics wind up protecting disease-causing germs. We have seen a tremendous increase in antibiotic-resistance in common food poisoning bacteria like Salmonella, but the problem is even worse than food-borne diseases. Bacteria also can rapidly transfer and spread antibiotic-resistance to other bacterial species. Therefore, diseases not even related to food become resistant to antibiotics, and hence much greater threats. For example, Staphylococci resistant to every available antibiotic have been isolated in recent years. Acquisition of antibiotic-resistance by bioterrorism weapons are also a concern. Use of antibiotics in animal feed, by selecting for antibiotic-resistant bacteria, is thus a global threat to human health. Major scientific and medical organizations have concluded that agricultural uses of antibiotics pose a threat to public health. We need prescriptions for these drugs, yet the animal-food industries use them casually. This irresponsible misuse of antibiotics is unilaterally disarming our species from our precious last line of defense, and devastating epidemics may be the legacy of hunger for inexpensive meat. Legislation is urgently needed to curb this practice.