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Structural organization and transcription of plant mitochondrial and chloroplast genomes.

Experimental evidence is presented showing that the plant mitochondrial and chloroplast genomes are multipartite and, that besides a large circular genomic DNA, they contain subgenomic minicircular and plasmid-like molecules. It is demonstrated that plant mitochondrial and chloroplast DNAs are packaged into deoxynucleoprotein fibrils comprising nucleosome-like and nucleomere-like globules; the fibrils form loops and rosette-like structures with central proteinaceous components. A similar structure is characteristic of the subgenomic DNAs. The basic proteins involved in the formation of nucleosome-like globules are quite different from the nuclear histones, indeed the basic proteins from plant mitochondria and chloroplasts are also distinct. Some of the basic proteins share common antigens with the E. coli HU protein. The genetic code for the mitochondrial and chloroplast genes is universal. The only codon now thought to be different from the universal in the mitochondrial genome is corrected during post-transcriptional mRNA editing. There are two hexanucleotides in the promoters of the chloroplast genes homologous to the sequences in -10 and -35 regions of the prokaryotic genes promoters requisite for transcription. Promoter sequences of the plant mitochondria genes responsible for transcription regulation were not identified. Immunoelectronmicroscopic evidence suggest that mitochondrial and chloroplast RNA polymerases have antigens in common with the beta-subunit of E. coli RNA polymerase. It is shown that the mitochondrial genes are intensely transcribed in the dark and repressed by illumination. Electron microscopy demonstrated that about 70% of plant mitochondria contain numerous RNA polymerase molecules in the dark, but this percentage falls to 10-15% after light exposure.

Chloroplasts↗

Identification of rare maternal copy number variants by genome-wide analysis of noninvasive prenatal screening data in 113,017 pregnant women.

OBJECTIVES: Knowledge of copy number variants (CNVs) is relevant to maternal and fetal health and can be obtained from noninvasive prenatal screening (NIPS) of pregnancy. However, genome-wide analysis of maternal CNVs using NIPS data has not been conducted in large populations. METHODS: For CNV analysis, the human genome was segmented into 10 kilobase pairs (Kb) bins, and the relative sequencing depth of each bin was calculated. The circular binary segmentation algorithm was used to estimate CNVs. Detected CNVs from two pregnancies of the same participant were compared to validate the reproducibility. All CNVs were merged into CNV regions (CNVRs) to evaluate their frequency, distributions, and relationship with disease-related genes and regions. RESULTS: In this study, 113,017 pregnant women were recruited. A total of 363,886 CNVs larger than 50 Kb were detected in 101,779 individuals and merged into 43,005 CNVRs. For evaluating the reproducibility of CNVs, 90.18% of deletions and 88.07% of duplications were consistent. In general, 78.13% of individuals carried CNVRs that overlapped protein-coding genes, while 14.76% overlapped OMIM genes. We detected 246 novel CNVRs, 134 (54.47%) involving protein-coding genes. For the perspective of maternal-fetal health, we identified 4,984 (4.41%) individuals as carriers of 5,243 CNVs containing known pathogenic or likely pathogenic regions, including 22q11.2 region and DMD gene.. CONCLUSIONS: NIPS sequencing data is a reliable source for maternal CNV detection. These CNVs constitute an integrate component in maternal-fetal health management.

Humans↗

A laterally interconnected neural architecture in MST accounts for psychophysical discrimination of complex motion patterns.

The complex patterns of visual motion formed across the retina during self-motion, often referred to as optic flow, provide a rich source of information describing our dynamic relationship within the environment. Psychophysical studies indicate the existence of specialized detectors for component motion patterns (radial, circular, planar) that are consistent with the visual motion properties of cells in the medial superior temporal area (MST) of nonhuman primates. Here we use computational modeling and psychophysics to investigate the structural and functional role of these specialized detectors in performing a graded motion pattern (GMP) discrimination task. In the psychophysical task perceptual discrimination varied significantly with the type of motion pattern presented, suggesting perceptual correlates to the preferred motion bias reported in MST. Simulated perceptual discrimination in a population of independent MST-like neural responses showed inconsistent psychophysical performance that varied as a function of the visual motion properties within the population code. Robust psychophysical performance was achieved by fully interconnecting neural populations such that they inhibited nonpreferred units. Taken together, these results suggest that robust processing of the complex motion patterns associated with self-motion and optic flow may be mediated by an inhibitory structure of neural interactions in MST.

Animals↗

Dependence of overall correction factor of a cylindrical ionization chamber on field size and depth in medium-energy x-ray beams.

In this paper we examine the depth and field size dependence of the overall correction factor kch for in-phantom dose determinations in orthovoltage x-ray beams. The overall correction factor is considered to be composed of three contributions, i.e., (1) a contribution from the angular dependence of the chamber response free-in-air, derived based on the measured directional response of the NE2571 for different energies combined with Monte Carlo calculations; (2) a displacement effect and (3) a stem effect, both calculated using the Monte Carlo method for different field sizes and depths. The results show a variation of, at most, 2.2% at the lowest photon energies (29.8-keV average photon energy) when going from 2 cm to 5 cm for a small circular 20-cm2 field. In the medium-energy range (> or = 100 kV), variations are limited to, at most, 1.5% for 120 kV-150 kV when comparing the most extreme variations in field size and depth (i.e., 2-cm depth; 20-cm2 area compared to 5 cm depth; 200-cm2 area). Depth variations most importantly affect the overall correction factor by hardening of the photon fluence spectrum, whereas field diameter variations affect the factor by increase or decrease of contributions of photon scattering. The work shows that taking into account the uncertainties adopted in the recent review of data and methods recommended in the IAEA code of practice, the dependence of the overall correction factor on depth and field size is insignificant for the radiation qualities between 100 kV (HVL 0.17-mm Cu, average energy: 52 keV) and 280 kV (HVL 3.41-mm Cu, average energy: 144 keV).

Calorimetry↗

Genetic transformation of Streptococcus pneumoniae by heterologous plasmid deoxyribonucleic acid.

A number of heterologous plasmid deoxyribonucleic acids (DNAs) coding for erythromycin, tylosin, lincomycin, tetracycline, or chloramphenicol resistance have been introduced into Streptococcus pneumoniae via genetic transformation with frequencies that varied between 10(-5) to as high as 5 x 10(-1) per colony-forming unit. Transformation with plasmid DNA required pneumococcal competence, was competed by chromosomal DNA, and showed a saturation at about 0.5 micrograms/ml (with a recipient population of 3 x 10(7) colony-forming units of competent cells per ml). Plasmid transformation did not occur with a recipient strain, 410, defective in endonuclease I activity and in chromosomal genetic transformation. All erythromycin-resistant transformants examined contained covalently closed circular DNA with the same electrophoretic mobility on agarose gels as the donor DNAs, and when examined in detail the plasmid reisolated from the transformants had the same restriction patterns and the same specific transforming activity as the donor DNA. In the cases of two plasmids examined in detail--pAM77 and pSA5700 Lc9--most of the transforming activity was associated with DNA monomers; DNA multimers present in pSA5700 Lc9 also had biological activity. An unexpected finding was the demonstration of transformation (2 x 10(-5) per colony-forming unit) with plasmid DNAs linearized by treatment with S1 nuclease or with restriction endonucleases.

Bacillus subtilis↗

Structure and activity of granulocyte colony-stimulating factor derived from CHO cells containing cDNA coding for alternatively spliced sequences.

Two different cDNAs have been isolated, coding for two forms of granulocyte colony-stimulating factor (G-CSF): one for a polypeptide of 174 amino acids and the other for a polypeptide of 177 amino acids. In this paper, we have expressed these two forms in Chinese hamster ovary cells and characterized the purified proteins for activity and conformation. In vitro mitogenic assay showed a 50-fold lower activity for the 177 form than for the 174 form. In vitro receptor binding assay showed that binding of the 177 form to the purified extracellular domain of G-CSF receptor was also diminished, while the 174 form complexed with the receptor. Circular dichroic spectra showed that both forms are similar in the secondary structure, but are slightly different in the tertiary structure. Infrared spectra also showed a slight difference between the two forms. Both techniques also demonstrated differences in stability; i.e., the 174 form is more stable than the 177 form during storage or against heat denaturation.

Alternative Splicing↗

Effect of tandem rare codon substitution and vector-host combinations on the expression of the EBV gp110 C-terminal domain in Escherichia coli.

Gp110 of Epstein-Barr virus (EBV) is a glycoprotein that functions exclusively during the assembly of EBV nucleocapsid and the release of infectious EBV. Its C-terminal tail domain (gp110 CTD) is essential for gp110's function and may provide signals that are responsible for the assembly and release of EBV. In the present study, to get large amounts of gp110 CTD for structural analysis, the effects of vector system, codon usage, and host strain on expression levels of gp110 CTD in Escherichia coli have been investigated. The coding region of gp110 CTD (11 kDa) was subcloned into the expression vectors pSE 280, pET-15b, pET-29a, pMAL-c2x, and pGEX-4T-1. Except the pMAL-c2x construct, all the others failed to express detectable amounts of recombinant gp110 CTD. Substituting a tandem rare AGA (Arg) codon with a synonymous CGC (Arg) codon facilitated expression of the recombinant protein, while a protease-deficient host E. coli strain helped in the accumulation of a soluble form of gp110 CTD fusion. The secondary structures of the obtained recombinant gp110 CTD purified from soluble extracts and inclusion bodies were compared using circular dichroism analysis. In aqueous solutions, both samples equally adopt a mixed alpha-helix and beta-sheet conformation as well as a partly unordered structure. Notably, in the membrane-mimicking environments the helical propensity of gp110 CTD increased up to the previously predicted level based on its sequence, suggesting that gp110 CTD may fold into a more stable conformation through interactions with the cell membrane.

Circular Dichroism↗

Chromosome 3p14 homozygous deletions and sequence analysis of FRA3B.

Loss of heterozygosity (LOH) involving 3p occurs in many carcinomas but is complicated by the identification of four distinct homozygous deletion regions. One putative target, 3p14.2, contains the common fragile site, FRA3B, a hereditary renal carcinoma-associated 3;8 translocation and the candidate tumor suppressor gene, FHIT. Using a approximately 300 kb comsid/lambda contig, we identified homozygous deletions in cervix, breast, lung and colorectal carcinoma cell lines. The smallest deletion (CC19) was shown not to involve FHIT coding exons and no DNA sequence alterations were present in the transcript. We also detected discontinuous deletions as well as deletions in non-tumor DNAs, suggesting that FHIT is not a selective target. Further, we demonstrate that some reported FHIT aberrations represent normal splicing variation. DNA sequence analysis of 110 kb demonstrated that the region is high in A-T content, LINEs and MER repeats, whereas Alu elements are reduced. We note an intriguing similarity in repeat sequence composition between FRA3B and a 152 kb segment from the Fragile-X region. We also identified similarity between a FRA3B segment and a small polydispersed circular DNA. In contrast to the selective loss of a tumor suppressor gene, we propose an alternative hypothesis, that some putative targets including FRA3B may undergo loss as a consequence of genomic instability. This instability is not due to DNA mismatch repair deficiency, but may correlate in part with p53 inactivation.

Acid Anhydride Hydrolases↗

Complete nucleotide sequence of minicircle kinetoplast DNA from Trypanosoma equiperdum.

The kinetoplast DNA of Trypanosoma equiperdum is composed of about 3000 supercoiled minicircles of 1000 base pairs and about 50 supercoiled maxicircles of 23,000 base pairs topologically interlocked so as to form a compact network. Minicircles of T. equiperdum, which are homogeneous in base sequence, were purified by equilibrium CsCl centrifugation and used as starting material for DNA sequence analysis. One minicircle is composed of 1012 base pairs and has an adenine.thymine base pair content of 72.8%. The termination codons are uniformly distributed along the molecule and restrict the coding potentiality of the molecule to oligopeptides of about 20 amino acids. The molecule contains three dyad symmetries and a sequence of 12 nucleotides is repeated six times. We also noted the presence of a region of about 130 base pairs that is almost perfectly homologous with that of the minicircles from the closely related species T. brucei.

Animals↗

DNA sequence and transcription of a DNA minicircle isolated from male-fertile sugar beet mitochondria.

A minicircle consisting of 1440 bp was isolated from male fertile sugar beet mitochondria. The entire nucleotide sequence of the minicircle was determined. There are several stop codons in all three reading frames and it is therefore unlikely that the minicircle codes for a polypeptide product. However, the minicircle is transcribed yielding a major RNA product consisting of 850 bases in addition to two very minor RNA products of 1100 and 950 bases, respectively. All three RNA products are transcribed from the same strand. The starting point for transcription of the major RNA product was determined by S1 nuclease protection analysis.

Base Composition↗

Isolation, characterization, and chromosomal localization of human brain cDNA clones coding for the precursor of the amyloid of brain in Alzheimer's disease, Down's syndrome and aging.

Four clones have been isolated from the adult human brain cDNA library using an oligonucleotide probe corresponding to the first 20 amino acids of the brain amyloid polypeptide. The open reading frame of the sequenced clone coded for the known full amino acid sequence of the brain amyloid polypeptide. The 3 kb messenger RNA has been detected in a variety of tissues from human and many nonhuman species. The gene is highly conserved in evolution and has been mapped on human chromosome 21.

Aging↗

Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase Lck.

Herpesvirus saimiri codes for a tyrosine kinase interacting protein (Tip) that interacts with both the SH3 domain and the kinase domain of the T-cell-specific tyrosine kinase Lck via two separate motifs. The activation of Lck by Tip is considered as a key event in the transformation of human T-lymphocytes during herpesviral infection. We investigated the interaction of proline-rich Tip peptides with the LckSH3 domain starting with the structural characterization of the unbound interaction partners. The solution structure of the LckSH3 was determined by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy using 44 residual dipolar couplings in addition to the conventional experimental restraints. Circular dichroism spectroscopy proved that the polyproline helix of Tip is already formed prior to SH3 binding and is conformationally stable. NMR titration experiments point out three major regions of the Tip-Lck interaction comprising the RT loop, the n-src loop, and a helical turn preceding the last strand of the beta-sheet. Further changes of the chemical shifts were observed for the N- and C-terminal beta-strands of the SH3 domain, indicating additional contacts outside the proline-rich segment or subtle structural rearrangements transmitted from the binding site of the proline helix. Fluorescence spectroscopy shows that Tip binds to the SH3 domains of several Src kinases (Lck, Hck, Lyn, Src, Fyn, Yes), exhibiting the highest affinities for Lyn, Hck, and Lck.

Amino Acid Sequence↗

Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization.

The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24 h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40-50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kb Hind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells of Escherichia coli, and colonies were recovered that carried pGEM with Hind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transform E. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning into E. coli.

Chloramphenicol O-Acetyltransferase↗

Relating cone signals to color appearance: failure of monotonicity in yellow/blue.

Observers performed red-green and yellow-blue hue cancellation tasks for a 0.8-deg circular test field on a dark surround, by manipulating the excitation level of one cone class while the other two classes were held constant. The results of the red-green judgments conformed to classical opponent color theory in that both L- and S-cone excitation levels were antagonistic to M-cone signals. The yellow-blue judgments revealed a nonmonotonic nonlinearity in which the S-cone signal could act either antagonistically or synergistically with M- and L-cone signals. These results demonstrate that fixed hue sensations should not be associated with a given class of cone, even at the level of opponent neural coding.

Color Perception↗

Translocation of an ampicillin resistance determinant within an R-factor aggregate in Salmonella panama.

The molecular properties of the plasmids of a natural isolate of Salmonella panama have been studied. This strain, Sp477, harbours 5 different plasmids: the conjugative plasmid pRI477TF (molecular weight 20 megadaltons), the two non-conjugative plasmids, pRI477A and pRI477S, coding for ampicillin and streptomycin plus sulfonamide resistance respectively (molecular weights of both 5.6 megadaltons) and two cryptic plasmids with molecular weights of 1.0 and 2.7, megadaltons respectively. After conjugal transfer to Escherichia coli the ampicillin resistance determinant was frequently found to be integrated into pRI477TF or pRI477S. The translocatable sequence on pRI477A, designated as Tn901, resembles the TnA sublcass transposon TnA(1).

Ampicillin↗

A Sequential Scenario for the Origin of Biological Chirality

A sequential model is proposed regarding the origin of biological chirality. Three major stages are presumed: a symmetry breaking (prebiotic chiral disruption in enantiomeric mixtures of monomers), a chiral amplification (prebiotic increase of the chiral character of the monomers affected first by the symmetry breaking), and a chiral expansion (proto biological increase of the chiral character and spread of the chirality to molecules which were less affected by prebiotic chiralizations). As a symmetry-breaking mechanism, the model proposed by Deutsch (1991) is used, which involves a dissymmetric exposure of amino acids (AA) to ultraviolet circularly polarized light (UV-CPL) on evaporative seashores. It is presumed that the chiral amplification, up to a protobiologic significance, was influenced by a periodic overlapping of two abiotic events, a synchronization between tidal-based hydrous-anhydrous cycles, and littoral asymmetric photolysis cycles. This long-term astronomic asymmetry acted around 3.8-4.2 billion years ago and was unique to the Earth in our solar system. It is also presumed that the abiotic symmetry breaking is heterogenous, that only a few l-AAs were used in the beginning, and that the chirality expanded later to all 20 AAs based on a coevolutionary strategy of the genetic code and on a physiological relationship between AAs. In this scenario the d-chirality of pentoses in polynucleotides was attributed to both d-pentose/l-AA relationships and to a structural evolution.

Journal Article↗

Structural and biosensor analyses of a synthetic biotinylated peptide probe for the isolation of adenomatous polyposis coli tumor suppressor protein complexes.

Large numbers of colon tumors stem from mutations in the gene coding for the production of the adenomatous polyposis coli (APC) tumor suppressor protein. This protein contains a coiled-coil N-terminal domain that is known to be responsible for homodimerization. Previous work by others has led to the design of a specific 54-residue anti-APC peptide (anti-APCp1) that dimerizes preferentially with this domain. We have undertaken the chemical synthesis of a modified form of this peptide (anti-APCp2) that bears a biotin moiety at its N-terminus for use in subsequent ligand-binding analysis studies. The peptide was subjected to comprehensive chemical characterization to confirm its purity. Secondary structural analysis by circular dichroism spectroscopy and Fourier transform infrared spectroscopy indicated that the peptide could assume a wide range of potential conformations, depending upon the precise microenvironment. Significantly, a stable alpha-helical structure was generated when the solvent conditions supported intramolecular salt-bridge formation along the helix barrel. The biotinylated anti-APCp2 was immobilized onto a streptavidin sensor surface, in a specific orientation leaving all amino acids available to form a coiled structure. In one experiment, injection of colonic cell lysate extracts (LIM1215) onto a size-exclusion column resulted in the isolation of a high molecular mass protein peak (> 600 kDa) that reacted specifically with the immobilized anti-APCp2 on the biosensor surface. In another experiment, a high molecular mass protein (M(r) > 250 kDa on SDS-PAGE) could be specifically immunoprecipitated from this peak using either the anti-APCp2 peptide or an anti-APC polyclonal antibody. This demonstrates the specific interaction between the anti-APCp2 peptide and native APC and highlights the potential use of the former peptide in a multidimensional micropreparative chromatographic/biosensor/proteomic protocol for the purification of APC alone and APC complexed with different biopolymers in various cell lines, and stages of tumor development.

Adenomatous Polyposis Coli↗

Viroids and prions.

Viroids are small "naked" infectious RNA molecules that are pathogens of higher plants. The potato spindle tuber viroid (PSTV) is composed of a covalently closed circular RNA molecule containing 359 ribonucleotides. The properties of PSTV were compared with those of the scrapie agent, which causes a degenerative neurological disease in animals. PSTV was inactivated by ribonuclease digestion, psoralen photoadduct formation, Zn2+ -catalyzed hydrolysis, and chemical modification with NH2OH. The scrapie agent resisted inactivation by these procedures, which modify nucleic acids. The scrapie agent was inactivated by proteinase K and trypsin digestion, chemical modification with diethylpyrocarbonate, and by exposure to phenol, NaDodSO4, KSCN, or urea. PSTV resisted inactivation by these procedures, which modify proteins. Earlier evidence suggested that the scrapie agent is smaller than PSTV. Its small size seems to preclude the presence of a genome coding for the protein(s) of a putative capsid. The properties of the scrapie agent distinguish it from both viroids and viruses and have prompted the introduction of the term "prion" to denote a small proteinaceous infectious particle that resists inactivation by procedures that modify nucleic acids.

Diethyl Pyrocarbonate↗