Chromosomal localization of the human interleukin 1 alpha (IL-1 alpha) gene

The human interleukin 1 alpha gene was assigned to chromosome 2 using Southern transfer analysis of human-rodent somatic cell hybrid DNAs. The gene was regionally localized to 2q12-21 using in situ hybridization to metaphase chromosomes. These results indicate that the IL-1 alpha gene maps to the same general region on the long arm of chromosome 2 as the IL-1 beta gene, which has been previously assigned.     

Chromosomal localization of the human interleukin 1α (IL-1α) gene

The human interleukin 1α gene was assigned to chromosome 2 using Southern transfer analysis of human-rodent somatic cell hybrid DNAs. The gene was regionally localized to 2q12–21 using in situ hybridization to metaphase chromosomes. These results indicate that the IL-1α gene maps to the same general region on the long arm of chromosome 2 as the IL-1β gene, which has been previously assigned.     

The human myelin-basic-protein gene: chromosomal localization and RFLP analysis.

With a human myelin-basic-protein (MBP) cDNA used as a probe, the human MBP gene has been mapped to chromosome region 18q22-q23 by a combination of Southern hybridization to a panel of somatic-cell hybrid DNAs and in situ hybridization to metaphase chromosomes. Restriction-fragment-length polymorphisms (RFLPs) have also been identified with this probe in human DNA, by means of the restriction enzymes BamHI, PvuII, and PstI. In studies of informative families, the alleles of the BamHI and PvuII polymorphisms have been shown to segregate as Mendelian traits.     

Analysis of intrasugar interproton NOESY cross-peaks as an aid to determine sugar geometries in DNA fragments

A systematic analysis of the conformation of deoxyribofuranose rings in DNA fragments has been described using two-dimensional nuclear Overhauser effect spectroscopy (2D NOESY). The approach is based on the interpretation of the intrasugar proton-proton distances which can be estimated using a low-mixing-time pure-absorption mode w1-scaled NOESY spectrum. The experimental distances are compared with the theoretical values calculated as a function of pseudorotation phase angle (P) describing the sugar geometries. The approach can be used as a complementary aid to J couplings for establishing sugar conformations in individual nucleotide units of DNA fragments. Using this strategy on d-ACATCGATGT, we observed that individual nucleotides exhibit O4'-endo sugar pucker. The results rule out possibilities of the existence of a fast equilibrium (on the NMR time scale) between C2'-endo (or S-domain) and C3'-endo (or N-domain) sugar puckers.     

Interaction of thiocyanate with horseradish peroxidase. 1H and 15N nuclear magnetic resonance studies

Interaction of thiocyanate with horseradish peroxidase (HRP) was investigated by relaxation rate measurements (at 50.68 MHz) of the 15N resonance of thiocyanate nitrogen and by following the hyperfine shifted ring methyl proton resonances (at 500 MHz) of the heme group of SCN-.HRP solutions. At pH 4.0, the apparent dissociation constant (KD) for thiocyanate binding to HRP was deduced to be 158 mM from the relaxation rate measurements. Chemical shift changes of 1- and 8-ring methyl proton resonances in the presence of various amounts of thiocyanate at pH 4.0 yielded KD values of 166 and 136 mM, respectively. From the pH dependence of KD and the 15N resonance line width, it was observed that thiocyanate binds to HRP only under acidic conditions (pH less than 6). The binding was found to be facilitated by protonation of an acid group on the enzyme with pKa 4.0. The pH dependence of the 15N line width as well as the apparent dissociation constant were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate in deprotonated ionic form binds to the enzyme in protonated acidic form. The KD for thiocyanate binding to HRP was also evaluated in the presence of an excess of exogenous substrates such as resorcinol, cyanide, and iodide ions. It was found that the presence of cyanide (which binds to heme iron at the sixth coordination position) and resorcinol did not have any effect on the binding of thiocyanate, indicating that the binding site of the thiocyanate ion is located away from the ferric center as well as from the aromatic donor binding site. The KD in the presence of iodide, however, showed that iodide competes with thiocyanate for binding at the same site. The distance of the bound thiocyanate ion from the ferric center was deduced from the 15N relaxation time measurements and was found to be a 6.8 A. From the distance as well as the change in the chemical shifts and line width of 1- and 8-methyl proton resonances, it is suggested that the binding site of thiocyanate may be located near heme, placed symmetrically with respect to 1- and 8-methyl groups of the heme of HRP. Similarity in the modes of binding of iodide and thiocyanate suggests that the oxidation of thiocyanate ion by H2O2 may also proceed via the two-electron transfer pathway under acidic conditions, as is the case for iodide.     

All-Cause Mortality of Low Birthweight Infants in Infancy,Childhood, and Adolescence: Population Study of England and Wales

BackgroundLow birthweight (LBW) is associated with increased mortality in infancy, but its association with mortality in later childhood and adolescence is less clear. We investigated the association between birthweight and all-cause mortality and identified major causes of mortality for different birthweight groups.ConclusionsLBW is associated with infant and later child and adolescent mortality, with perinatal factors and congenital malformations explaining many of the deaths. By understanding and ameliorating the influences of upstream exposures such as maternal smoking and deprivation, later mortality can be decreased by reducing the delivery of vulnerable infants with LBW.     

An approach to delineate primers for a group of poorly conserved sequences incorporating the common motif region

Glutathione synthetase (gshB) has previously been reported to confer tolerance to acidic soil condition in Rhizobium species. Cloning the gene coding for this enzyme necessitates the designing of proper primer sets which in turn depends on the identification of high quality sequence similarity in multiple global alignments. In this experiment, a group of homologous gene sequences related to gshB gene (accession no: gi-86355669:327589-328536) of Rhizobium etli CFN 42, were extracted from NCBI nucleotide sequence databases using BLASTN and were analyzed for designing degenerate primers. However, the T-coffee multiple global alignment results did not show any block of conserved region for the above sequence set to design the primers. Therefore, we attempted to identify the location of common motif region based on multiple local alignments employing the MEME algorithm supported with MAST and Primer3. The results revealed some common motif regions that enabled us to design the primer sets for related gshB gene sequences. The result will be validated in wet lab.     

Production of 7-O-methyl aromadendrin, a medicinally valuable flavonoid, in Escherichia coli

7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important flavonoid-glycosides found in several plants, such as Populus alba and Eucalyptus maculata, with various medicinal applications. To produce such valuable natural flavonoids in large quantity, an Escherichia coli cell factory has been developed to employ various plant biosynthetic pathways. Here, we report the generation of 7-OMA from its precursor, p-coumaric acid, in E. coli for the first time. Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feeding p-coumaric acid and reconstructing the plant biosynthetic pathway by introducing the following structural genes: 4-coumarate-coenzyme A (CoA) ligase from Petroselinum crispum, chalcone synthase from Petunia hybrida, and chalcone isomerase from Medicago sativa. In order to increase the availability of malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α and β subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinica were also introduced. Thus, produced NRN was hydroxylated at position 3 by flavanone-3-hydroxylase from Arabidopsis thaliana, which was further methylated at position 7 to produce 7-OMA in the presence of 7-O-methyltransferase from Streptomyces avermitilis. Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as intermediate products. Overexpression of the genes for flavanone biosynthesis and modification pathways, along with malonyl-CoA overproduction in E. coli, produced 2.7 mg/liter (8.9 μM) 7-OMA upon supplementation with 500 μM p-coumaric acid in 24 h, whereas the strain expressing only the flavanone modification enzymes yielded 30 mg/liter (99.2 μM) 7-OMA from 500 μM NRN in 24 h.     

Noise suppression in stochastic genetic circuits using PID controllers

Inside individual cells, protein population counts are subject to molecular noise due to low copy numbers and the inherent probabilistic nature of biochemical processes. We investigate the effectiveness of proportional, integral and derivative (PID) based feedback controllers to suppress protein count fluctuations originating from two noise sources: bursty expression of the protein, and external disturbance in protein synthesis. Designs of biochemical reactions that function as PID controllers are discussed, with particular focus on individual controllers separately, and the corresponding closed-loop system is analyzed for stochastic controller realizations. Our results show that proportional controllers are effective in buffering protein copy number fluctuations from both noise sources, but this noise suppression comes at the cost of reduced static sensitivity of the output to the input signal. In contrast, integral feedback has no effect on the protein noise level from stochastic expression, but significantly minimizes the impact of external disturbances, particularly when the disturbance comes at low frequencies. Counter-intuitively, integral feedback is found to amplify external disturbances at intermediate frequencies. Next, we discuss the design of a coupled feedforward-feedback biochemical circuit that approximately functions as a derivate controller. Analysis using both analytical methods and Monte Carlo simulations reveals that this derivative controller effectively buffers output fluctuations from bursty stochastic expression, while maintaining the static input-output sensitivity of the open-loop system. In summary, this study provides a systematic stochastic analysis of biochemical controllers, and paves the way for their synthetic design and implementation to minimize deleterious fluctuations in gene product levels.