Functional homology between the sequence-specific DNA-binding proteins nuclear factor I from HeLa cells and the TGGCA protein from chicken liver.

Nuclear factor I from HeLa cells, a protein with enhancing function in adenovirus DNA replication, and the chicken TGGCA protein are specific DNA-binding proteins that were first detected by independent methods and that appeared to have similar DNA sequence specificity. To test whether they are homologous proteins from different species we have compared (i) their DNA binding properties and (ii) their function in reconstituted adenovirus DNA replication systems. Using deletion and substitution mutants derived from the DNA binding site on the adenovirus 2 inverted terminal repeat, it was found that the two proteins protect the same 24-nucleotide region of both strands against DNase I digestion and that they have identical minimal recognition sequences of 15 bp containing dyad symmetry. Like nuclear factor I, the TGGCA protein enhances the initiation reaction of adenovirus 2 DNA replication in vitro in a DNA recognition site-dependent manner.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Bacteria-induced haemolymph proteins of Manduca sexta pupae and larvae

The haemolymph of Manduca sexta larvae and pupae has been analyzed for proteins potentially associated with the bacterial defence response of the insect. Five proteins, M13, M18, M20, M23, and M24 in pupae, and M4, M11, M13, M18 and M23 in larvae, are induced by the injection of bacteria into the haemolymph. Proteins M4 and M11 are always present at high levels in uninjected pupae. Proteins M20 and M24 could not be induced in larvae. These proteins, as well as those not showing a response to bacterial challenge or injury, were also analyzed for presence of disulphide bonds and carbohydrate moieties, and their apparent molecular weights determined.     

Impaired incision of ultraviolet-irradiated deoxyribonucleic acid in uvrC mutants of Escherichia coli.

The production of single-strand breaks in the deoxyribonucleic acid of irradiated uvrC mutants of Escherichia coli K-12 was studied both in vivo and in vitro. In vivo, uvrC mutants displayed a slow accumulation of breaks after irradiation, and in this respect appeared different from uvrA mutants, in which very few breaks could be detected. The breakage observed in uvrC mutants differed from that observed in wild-type strains in both the slow rate of break accumulation and the very limited dose response. The behavior of the uvrC lig-7(Ts) double mutant was shown not to be consistent with the suggestion of ligase reversal as the explanation for the lower rate and limited dose response of break formation observed in ultraviolet-irradiated uvrC mutants in vivo. Rather, there appeared to be a real defect in incision. In toluene-treated cells, we studied the effect of the ligase inhibitor nicotinamide mononucleotide on strand incision. Whereas uvrC mutants displayed more strand breakage in the presence of this inhibitor, the same amount of breakage was seen in uvrA mutants, and as such the breakage could be judged as not due to the main excision repair pathway. Experiments using a cell-free system comprising the partially purified uvr+ gene products demonstrated clearly that there is a requirement for the uvrC+ gene product for strand incision. We suggest that in vivo in the absence of the uvrC+ gene product, a partial analog of this protein may allow some abnormal incision.     

Interaction of the UvrABC endonuclease with DNA containing a psoralen monoadduct or cross-link. Differential effects of superhelical density and comparison of preincision complexes.

The effect of negative supercoiling on UvrABC incision of covalently closed duplex DNA circles containing either a furan-side monoadduct or a cross-link of 4'-hydroxymethyl-4,5',8-trimethylpsoralen at a unique site was examined. The rate of UvrABC incision of these DNA substrates was measured as a function of superhelical density, sigma, for values of sigma between 0 and -0.050. The monoadducted DNA substrate was incised at close to the maximum rate at all superhelical densities, with only a slight stimulation of activity between sigma = 0 and -0.035. In contrast, efficient UvrABC incision of the cross-linked DNA substrate required the DNA to be underwound, and activity showed a linear dependence on superhelical density up to sigma = -0.035. DNase I protection studies show that in the presence of both UvrA and UvrB a protein complex binds to the site of a psoralen monoadduct or cross-link in linear DNA. This UvrA-UvrB-dependent complex binds with similar affinity to both the monoadducted and the cross-linked DNA helices. However, differences in the DNase I footprint on these two DNA substrates indicate that the interaction of this protein complex is different at these two lesions. The addition of UvrC to linear DNA molecules that are saturated at the site of the lesion with the UvrA-UvrB-dependent complex resulted in efficient nicking of the monoadducted DNA, but not the cross-linked DNA. Thus, the properties of a DNA lesion site that lead to UvrAB recognition and binding are not necessarily sufficient to allow incision when all three Uvr subunits are present. We propose that after recognition and binding of a lesion site by the UvrAB complex and prior to incision, the damaged DNA helix undergoes a conformational change such as unwinding or melting that is induced by the lesion-bound Uvr complex.     

Breeding soundness and sex drive by breed and age in beef bulls used for natural mating

A total of 92 range beef bulls (Hereford = 60; Angus = 32) were given a breeding soundness examination (BSE) and two assessments for sex drive prior to their use in 23 breeding trials employing estrous synchronized females. Bulls were in three age groups: yearlings (n=29), two year olds (n=36), and three year olds and older (n=27). All yearling bulls were virgins, but the majority of the older bulls had previous mating experience. Angus bulls were superior (P<0.01) to Herefords in spermatozoal morphology and BSE score. Scrotal circumference increased with age beyond two years in Angus bulls but not in Herefords. Spermatozoal abnormalities generally decreased with age. BSE scores did not differ significantly among age groups. Apart from number of mounts, measures of sex drive did not differ with age or breed of bulls. This represents qualified justification for the current practice of using the same sex-drive assessment procedures for Bos taurus bulls of various ages and breeds.     

Nuclear grooves in the aspiration cytology of papillary carcinoma of the thyroid

Nuclear grooving has recently been shown to be a useful morphologic feature in the diagnosis of papillary carcinoma of the thyroid in tissue sections and imprint smears. In order to assess the diagnostic value of nuclear grooving in cytologic specimens, the presence of this feature was evaluated in fine needle aspirates from 20 papillary carcinomas of the thyroid, 10 follicular adenomas, 3 follicular carcinomas, 1 medullary carcinoma, 10 nodular goiters and 4 cases of Hashimoto's thyroiditis. In each case, 30 random high-power fields (HPFs), or all fields in less cellular smears, were examined, and the percentage of the fields in which nuclear grooving could be seen was recorded. Seventeen of 20 papillary carcinomas (85%) showed nuclear grooves in more than 25% of the HPFs examined; in the remaining three cases, grooves were observed in less than 25% of the HPFs. In control cases (all other thyroid lesions), nuclear grooves either were absent or were present in less than 25% of the HPFs examined. These findings suggest that nuclear grooving, when seen in abundance, can be considered a reliable criterion for the diagnosis of papillary carcinoma in fine needle aspiration cytology of the thyroid. The presence of occasional grooves, however, should be regarded as a nonspecific finding.     

Diabetes-like action of intermittent fasting on sarcoplasmic reticulum Ca2+-pump ATPase and myosin isoenzymes can be prevented by sucrose

Experimental diabetes results in a reduction of the sarcoplasmic reticulum (SR) Ca2+-stimulated ATPase activity and a redirection of myosin isoenzymes from V1 to V3. Similar, but less pronounced, changes were induced by subjecting rats to intermittent fasting for 6 weeks. Low amounts of sucrose (0.8%) in the drinking water prevented the subcellular changes in fasted rats; however, sucrose neither affected the levels of plasma thyroid hormones nor normalized the reduced body weight. Plasma glucose was lowered without any changes in plasma insulin in the fasted rats receiving sucrose; this suggested an enhanced peripheral glucose utilization. Thus, the signals in the diabetic heart leading to changes in SR and myosin can be mimicked by intermittent fasting and seem to be linked to a shift in fuel utilization by the myocytes.     

Biotin-tubulin incorporates into kinetochore fiber microtubules during early but not late anaphase

The dynamic behavior of kinetochore fiber microtubules has been examined in PtK1 cells during anaphase of mitosis. Cells in anaphase were injected with biotin-tubulin and, at various intervals after injection, fixed for light or electron microscopic immunolocalization of biotin-tubulin-containing microtubules. When cells in early to mid anaphase were injected with biotin-tubulin and fixed 1-2 min later, fluorescence was observed throughout the spindle, including the region of the kinetochore fibers. Electron microscopy of early to mid anaphase cells, after processing with immunogold methods, revealed both labeled and unlabeled microtubules in the kinetochore fibers; some labeled microtubules contacted the kinetochores. When late anaphase cells were injected with biotin-tubulin, and fixed a few minutes later, little fluorescence was observed in the kinetochore fibers. Electron microscopy confirmed that kinetochore fibers in late anaphase cells were refractory to tubulin incorporation. The results of these experiments demonstrate that the kinetochore fiber incorporates new microtubules during early anaphase but that this incorporation ceases in mid to late anaphase. Thus, microtubule turnover within the kinetochore fiber does not abruptly cease at the onset of anaphase and anaphase kinetochore fiber microtubules are more dynamic than previously suspected.     

Molecular analysis of the rat MHC. II. Isolation of genes that map to the RT1.E-grc region

An initial mapping analysis of growth and reproduction complex (grc) and grc+ genomic DNA identified several restriction fragment length polymorphisms specific for the grc region of the MHC. To analyze further the genomic organization and structure of the grc, a cosmid library was constructed from a grc+-bearing strain (R21). One cosmid cluster, encompassing 41.4 kb of DNA, contained four, or possibly five, class I genes that mapped to the RT1.E-grc region Two unique non-class I fragments were isolated from certain cosmids within this cluster. These fragments were hybridized to genomic DNA derived from five rat strains (BIL/2, R18, R21, R22, and BIL/1), and the results showed that grc-bearing rats have a deletion of at least 3.1 kb of DNA in the region immediately adjacent to the MHC. The loss of the genes in this region is probably the cause of the growth and reproductive defects in these animals and probably also of their increased susceptibility to chemical carcinogens.