Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity.

The genetic code is degenerate, but alternative synonymous codons are generally not used with equal frequency. Since the pioneering work of Grantham's group it has been apparent that genes from one species often share similarities in codon frequency; under the "genome hypothesis" there is a species-specific pattern to codon usage. However, it has become clear that in most species there are also considerable differences among genes. Multivariate analyses have revealed that in each species so far examined there is a single major trend in codon usage among genes, usually from highly biased to more nearly even usage of synonymous codons. Thus, to represent the codon usage pattern of an organism it is not sufficient to sum over all genes as this conceals the underlying heterogeneity. Rather, it is necessary to describe the trend among genes seen in that species. We illustrate these trends for six species where codon usage has been examined in detail, by presenting the pooled codon usage for the 10% of genes at either end of the major trend. Closely-related organisms have similar patterns of codon usage, and so the six species in Table 1 are representative of wider groups. For example, with respect to codon usage, Salmonella typhimurium closely resembles E. coli, while all mammalian species so far examined (principally mouse, rat and cow) largely resemble humans.     

The muskrat in biomedical research

Muskrats are aquatic rodents of moderate size which are plentiful throughout North America, but are not used commonly in the laboratory. Recently, we tested the feasibility of muskrats as experimental models and have found them to be acquired and cared for easily in conventional laboratory animal facilities. Some of their natural characteristics and diseases are described. The husbandry techniques that we used are presented and form a base for the preparation of future guidelines for the maintenance and use of feral animals in research. The results of some initial experiments testing the muskrat's utility for investigations of cardiorespiratory control mechanisms also are presented. Our data show that even anesthetized muskrats possess brisk and dramatic cardiovascular and respiratory reflexes. Our findings that their brains possess the cytoarchitectural and myeloarchitectural features comparable to other mammals, combined with their relative uniformity in size, has allowed us to locate specific neuronal loci stereotaxically. We suggest that the muskrat be considered as an experimental animal model for studies of the neural control of cardiorespiratory systems.     

Hydrazine stress in the diabetic: ornithine decarboxylase activity

Streptozotocin-induced diabetes of 7 weeks duration increased male Sprague-Dawley rat kidney ornithine decarboxylase activity by 4.8-fold but did not affect the liver enzyme. Hydrazine treatment of 4 hr duration stimulated equally kidney ornithine decarboxylase activities of nondiabetic and diabetic rats. Hydrazine treatment increased liver ornithine decarboxylase activity in the nondiabetic rat but did not increase it in the diabetic rat. Since hydrazine stimulates ornithine decarboxylase activity prior to polyamine and protein syntheses, we speculate that the lack of hydrazine stimulation of ornithine decarboxylase in the diabetic liver may be related in part to the unrestrained gluconeogenesis and depressed Kreb's cycle activity: the latter being required for protein synthesis.     

Flow cytometric DNA content of fresh tumor specimens using keratin-antibody as second stain for two-parameter analysis.

Studies concerning flow cytometric assessed DNA content reveal problems in interpretating DNA histograms of tumor specimens. The main problems are histograms with a broad coefficient of variation in the G0/G1 fraction; a high G2M fraction and samples with a low percentage of tumor cells. Therefore, in the present study, 382 fresh tumor specimens of carcinomas were analysed routinely, double labeled with, on the one hand, propidium-iodide for assessing DNA content and, on the other, a monoclonal keratin-antibody for marking epithelial and tumor cells. Of the 311 tumor samples, using single parameter analysis 165 (54%) were classified as DNA aneuploid and 146 (46%) as DNA "euploid." By double parameter analysis, 224 (72%) samples were keratin positive and 87 (27%) keratin negative and, of the 224 keratin positive tumors, 175 (78%) were DNA aneuploid and 49 (22%) DNA euploid. The DNA histograms of single and double parameter analysis were compared and it was concluded that in 24 cases (11%) keratin labeling was necessary to recognize DNA aneuploidy. In another 23 (10%) cases, keratin labeling was helpful in assessing DNA aneuploidy. Finally when the results of the 311 samples were combined, 215 (68%) were scored as DNA aneuploid and 99 (32%) DNA euploid. Thus the overall gain in assessing DNA aneuploidy using the double labeling technique is 14%. In conclusion, it is shown that keratin labeling on fresh tumor cell suspensions of epithelial tumors is of additional value in establishing DNA content. Because single parameter DNA assessment is adequate in approximately 60% of the tested samples, the double labeling technique can be performed routinely, or after initial single parameter DNA assessment. Histograms having a broad CV and/or a high G2M are good candidates for the double labeling technique. Using this technique, DNA-content assessment becomes more reliable.     

A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.

We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.     

NCI-H716 cells as a model for endocrine differentiation in colorectal cancer

In colonic neoplasms, endocrine differentiation is encountered not only in carcinoid tumors but also in adenocarcinomas, where endocrine cells may represent a distinct line of differentiation in the tumor. The significance of endocrine differentiation in colorectal cancer is not well established, partly because of the paucity of tumor cell lines which can serve as a model for studying endocrine differentiation. In this report we describe the properties of NCI-H716 cells, a cell line derived from a poorly differentiated adenocarcinoma of the caecum, under various in vitro conditions and as xenografts in athymic mice. Phenotypical properties were immunohistochemically assessed using a panel of differentiation related antibodies, and also by Northern blot analysis and by electron microscopy. Receptors for biogenic amines and peptide hormones were analyzed by ligand binding assay. These studies show that:

Developmental changes in barrier web structure under different levels of predation risk inNephila clavipes (Araneae: Tetragnathidae)

Individuals of the orb-weaving spider Nephila clavipesbuild complex webs with a region used for prey capture, the orb, and tangle webs opposite either face, the barrier webs. Barrier webs have been hypothesized to serve a variety of functions, including predator defense, and the primary function of the barrier web should be reflected in the relative size of the barrier to the orb under varying conditions of foraging success and predation risk. To investigate the effects of predation pressure and foraging success on barrier web structure, I conducted a comparative study in three disjunct populations that differed in predation risk and foraging success. Although both the orb web and the barrier webs are silk, there was no indication of a foraging-defense trade-off. Barrier web structure did not change during seasonal shifts in orb web size related to changes in preycapture rate, and barrier web silk density and orb radius were positively correlated. The hypothesis that the construction of barrier webs is in part a response to predation pressure was supported. Barrier webs do deflect attacks by some predators, and barrier webs built by small spiders, suffering frequent predation attempts, had a higher silk density than barrier webs built by larger individuals. Additionally, barrier web complexity decreased at a later age in areas with higher predation risk.     

Enhanced production of cellulase usingAcidothermus cellulyticus in fed-batch culture

Summary Fed-batch fermentations of Acidothermus cellulolyticus utilizing mixtures of cellulose and sugars were investigated for potential improvements in cellulase enzyme production. In these fermentations, we combined cellulose from several sources with various simple sugars at selected concentrations. The best source of cellulose for cellulase production was found to be ball-milled Solka Floc at 15 g/l. Fed-batch fermentations with cellobiose and Solka Floc increased cell mass only slightly, but succeeded in significantly enhancing cellulase synthesis compared to batch conditions. Maximum cellulase activities obtained from fermentations initiated with 2.5 g cellobiose/l and 15 g Solka Floc/l were 0.187 units (U)/ml, achieved by continuous feeding to maintain <0.1 g cellobiose/l, and 0.215 U/ml using the same initial medium when 2.5 g cellobiose/l was step-fed after the sugar was nearly consumed. In batch, dual-substrate systems consisting of simple sugars with Solka Floc, substrate inhibition was evident in terms of specific growth rates, specific productivity values, and maximum enzyme yields. Limiting concentrations of glucose or sucrose at 5 g/l, and cellobiose at 2.5 g/l, in the presence of Solka Floc, yielded cellulase activities of 0.134, 0.159, and 0.164 U/ml, respectively. Offprint requests to: M. E. Himmel