Transduction of Cellular Sequence by a Human Immunodeficiency Virus Type 1-Derived Vector

During studies examining the rate of human immunodeficiency virus type 1 (HIV-1) mutation in a single cycle of replication, the 5' long terminal repeat of one progeny provirus was found to contain an insertion of 147 bp including an entire tRNA sequence as well as an additional 66 bp insertion of nonviral origin. Database searches revealed that 65 of 66 bp aligned with the human CpG island sequence found on chromosomes 6, 14, and 17. Therefore it seems probable that it is of human cellular sequence origin and was transduced by HIV-1. This is the first demonstration that HIV-1 can capture a cellular sequence. The site of integration of the parental provirus was mapped to chromosome 1p32.1. Sequence with homology to the transduced CpG island was not found on chromosome 1, suggesting that the transduced cellular sequence was not linked to the site of viral integration.     

Genome-Wide Expression Analysis in Fibroblast Cell Lines from Probands with Pallister Killian Syndrome

Pallister Killian syndrome (OMIM: # 601803) is a rare multisystem disorder typically caused by tissue limited mosaic tetrasomy of chromosome 12p (isochromosome 12p). The clinical manifestations of Pallister Killian syndrome are variable with the most common findings including craniofacial dysmorphia, hypotonia, cognitive impairment, hearing loss, skin pigmentary differences and epilepsy. Isochromosome 12p is identified primarily in skin fibroblast cultures and in chorionic villus and amniotic fluid cell samples and may be identified in blood lymphocytes during the neonatal and early childhood period. We performed genomic expression profiling correlated with interphase fluorescent in situ hybridization and single nucleotide polymorphism array quantification of degree of mosaicism in fibroblasts from 17 Caucasian probands with Pallister Killian syndrome and 9 healthy age, gender and ethnicity matched controls. We identified a characteristic profile of 354 (180 up- and 174 down-regulated) differentially expressed genes in Pallister Killian syndrome probands and supportive evidence for a Pallister Killian syndrome critical region on 12p13.31. The differentially expressed genes were enriched for developmentally important genes such as homeobox genes. Among the differentially expressed genes, we identified several genes whose misexpression may be associated with the clinical phenotype of Pallister Killian syndrome such as downregulation of ZFPM2, GATA6 and SOX9, and overexpression of IGFBP2.     

Tyrosine and tyrosinate fluorescence of pig intestinal Ca2+-binding protein.

The single tyrosine residue in both pig and cow intestinal Ca2+-binding proteins fluoresces at 303 nm although the crystal structure of the cow protein shows a hydrogen bond between the hydroxy group of the tyrosine and glutamate-38 [Szebenyi & Moffat (1986) J. Biol. Chem. 261, 8761-8777]. The latter interaction suggests that tyrosinate fluorescence should dominate the emission spectra of these proteins. A fluorescence difference spectrum, produced by subtracting the spectrum of free tyrosine from the spectrum of the protein, gives a peak at 334 nm due to ionized tyrosine. That this component of the emission spectrum is not due to a tryptophan-containing contaminant is shown by its elimination when the protein is denatured by guanidine and when glutamate-38 is protonated. We conclude that, in solution, the tyrosine residue in this protein interacts occasionally with glutamate-38 but that a permanent hydrogen bond is not formed.     

The apeE Gene of Salmonella typhimurium Encodes an Outer Membrane Esterase Not Present in Escherichia coli

Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine β-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674–680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C₆ to C₁₆ fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli.     

Uncapping of viral messenger RNA by phosphodiesterase of fibroblast plasma membranes

Summary Isolated plasma membranes from mouse fibroblast lines 3T3 and its tranformant SV-3T3 contain a phosphodiesterase (oligonucleotidase, E.C. 3.1.4.19; nucleotide pyrophosphatase, E.C. 3.6.1.9) that splits capped and methylated messenger RNA obtained from both reovirus and vesicular stomatitis virus. The isolated membranes are free of demonstrable ribonuclease activity and split the mRNA to produce 7-methyl guanosine diphosphate as a product. With ATP as substrate for the phosphodiesterase enzyme, the product is AMP. Synthetic caps, AMP, ADP and ATP, but not cyclic AMP, can compete with the substratep-nitrophenyl thymidilic acid. A possible regulatory role on messenger translation is proposed.     

The apeE gene of Salmonella enterica serovar Typhimurium is induced by phosphate limitation and regulated by phoBR

Mutations in apeR, a regulatory locus of the outer membrane esterase apeE from Salmonella enterica serovar Typhimurium, were shown to be alleles of the pstSCAB-phoU high-affinity phosphate transport operon. Expression of apeE was induced by phosphate limitation, and this induction required the phoBR phosphate regulatory system.     

Strain selection,medium development and scale-up of toyocamycin production by streptomyces chrestomyceticus

The batch productivity (Q _TM) of the production of the nucleoside antibiotic toyocamycin (TM) by Streptomyces chrestomyceticus was increased ten-fold by selection of a UV generated mutant, optimization of pH, increasing incubation temperature from 28 °C to 36 °C, and addition of soy oil. Initial high oxygen transfer rates stimulated Q _TM maxima two-fold. Antibiotic production by the mutant strain, U190, however, appeared more shear sensitive than the parent culture FCRF 341 with maximum antibiotic titer being inversely related to impellor tip velocity, T _v . For this reason, scale-up could not be done at constant P/V or constant volumetric oxygen transfer. Instead, programming of impeller speed was evaluated in order to maintain optimal impeller tip velocity during scale-up. It was found that a low constant T _v maintained in scale-up in geometrically similar vessels was most beneficial for duplication of optimal antibiotic productivity, Q _TM. Pilot fermentations (120 dm³ scale) were used to determine coefficients of Q _TM variation from oxygen uptake rate (OUR) and total CO₂ evolution data for monitoring of Q _TM variation during scale-up to the 12,000 dm³ scale. This technique allowed for on-line prediction of antibiotic titer and Q _TM from fermentor exhaust gas data.List of Symbols A scale constant - B shape constant - C location of maximum constant - D m impeller diameter (m) - H m liquid height (m) - OTR MmolO₂·(dm³)^–1min^–1 oxygen transfer rate - OUR MmolO₂·(dm³)^–1min^–1 oxygen uptake rate - PCV cm³ packed cell volume - P/V watts/dm³ volumetric power consumption - Q 1 · min^–1 corrected to standard conditions of temperature, pressure aeration rate - Q _TM g/(cm³ · h) or kg/(m³ · h) antibiotic productivity - T m tank diameter - T _mix s mixing time - T _v cm · s^–1 impeller tip velocity - TM g/cm³ Toyocamycin concentration - TNP Tricyclic nucleoside phosphate     

Interspecific Comparison of the Transformer Gene of Drosophila Reveals an Unusually High Degree of Evolutionary Divergence

The transformer (tra) gene of Drosophila melanogaster occupies an intermediate position in the regulatory pathway controlling all aspects of somatic sexual differentiation. The female-specific expression of this gene's function is regulated by the Sex lethal (Sxl) gene, through a mechanism involving sex-specific alternative splicing of tra pre-mRNA. The tra gene encodes a protein that is thought to act in conjunction with the transformer-2 (tra-2) gene product to control the sex-specific processing of doublesex (dsx) pre-mRNA. The bifunctional dsx gene carries out opposite functions in the two sexes, repressing female differentiation in males and repressing male differentiation in females. Here we report the results from an evolutionary approach to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interspecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event.     

Circadian Hematologic Time Structure in the Elderly

Twenty-three clinically healthy, diurnally active elderly subjects, 71 ± 5 years of age were studied over a 24-hr span (six samples). Complete blood counts and differential counts were done (Ortho ELT-8, Wright stained smears). The circadian rhythm parameters of the hematologic variables in the elderly subjects were compared with reference values obtained from a larger group of clinically healthy young adult and adult subjects studied independently. The data were analyzed by cosinor and the Bingham test. Circadian rhythms in the number of circulating formed elements in the peripheral blood persist in the aged. In comparison with the young adult, the elderly subjects show differences in the timing (phase advance) of the circadian rhythms in circulating neutrophil leukocytes and lymphocytes, a decrease in the circadian amplitude of circulating platelets, a decrease in circadian rhythm adjusted mean (mesor) in the red cell count, and in the neutrophil band forms.