Size and physical organization of chloroplast DNA from mustard (Sinapis alba L.)

Summary Intact chloroplast (cp)DNA from mustard cotyledons (Sinapis alba L.) was found by electron microscopy to be a uniform population of circular molecules with a contour length corresponding to 158 kilobase pairs. This size was confirmed by restriction endonuclease analysis. Nucleases SalI and XhoI each generate a small number of cpDNA fragments. The sizes of all fragments generated by each enzyme sum up to more than 150 kilobase pairs. Overlaps of SalI and XhoI fragments were determined by double digestion and triple digestion including SmaI. A physical map of mustard cpDNA with reference to all recognition sites for SalI and most sites for XhoI is presented. This map indicates that an inverted repeated sequence covers approximately 30% of the molecule and is interrupted by two unique sequence regions of different sizes.     

Structural characterization of glycolipids of rat small intestine having one to eight hexoses in a linear sequence

Eight different fractions containing glycolipids with 1 to 8 hexoses in a linear sequence were isolated from rat small intestine. The structure of the major components was established by mass spectrometry and proton nuclear magnetic resonance spectroscopy of the permethylated and permethylated-reduced (LiAlH₄) derivatives and by gas-liquid chromatography of degradation products of the native and permethylated or permethylated-reduced glycolipids. The major compounds were glucosylceramide, lactosylceramide, globotriaosylceramide, and a novel tetrahexosylceramide with the structure Gal α 1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer. In addition four minor compounds having five to eight hexoses were identified with the probable structures Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Galα1 → 3Galα1 → 3Galα1 → 3Galα1 → 4Galβ1 → 4Glcβ1 → 1Cer, Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer, and Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 3Gal1 → 4Gal1 → 4Glc1 → 1Cer. In the pentahexosylceramide fraction a novel fucolipid was also present having the probable structure Fucα1 → 2Galα1 → 3Galα1 → 4Galβ1 → 4Galβ1 → 1Cer. The lipophilic part of the glycolipids was composed of trihydroxy 18:0 and dihydroxy 18:1 long-chain bases in combination with nonhydroxy and hydroxy 16:0–24:0 fatty acids. Glycolipid studies of isolated mucosal epithelial cells and the nonepithelial intestinal residue revealed a specific cell distribution of these hexosyl compounds. The two major components, glucosylceramide and globotriaosylceramide, were mainly located in the epithelial cells together with small amounts of lactosylceramide and tetrahexosylceramide. The epithelial cells practically lacked however the penta- to octahexosylceramides. The nonepithelial residue contained all hexosyl compounds. The fucolipid was exclusively present in the epithelial cells.     

RAPID CHEMICAL DEHYDRATION OF PLANT MATERIAL FOR LIGHT AND ELECTRON MICROSCOPY WITH 2,2-DIMETHOXYPROPANE AND 2,2-DIETHOXYPROPANE

A rapid and simple procedure was used for chemical dehydration of plant tissue during sample preparation for light and electron microscopy. Chemically fixed tissues were washed with distilled water and then rapidly dehydrated with either 2,2-dimethoxypropane or 2,2-diethoxypropane for 15 minutes. Light microscopic observation of paraffin-embedded tissue or tissue embedded in Spurr's plastic showed excellent preservation. Electron microscopic examination of plastic-embedded tissue showed well maintained ultrastructural morphology. The dehydration procedure was also successfully applied to plant tissue destined for examination in a scanning electron microscope.     

Effect of genetic heterogeneity and assortative mating on linkage analysis: a simulation study.

Linkage studies of complex genetic traits raise questions about the effects of genetic heterogeneity and assortative mating on linkage analysis. To further understand these problems, I have simulated and analyzed family data for a complex genetic disease in which disease phenotype is determined by two unlinked disease loci. Two models were studied, a two-locus threshold model and a two-locus heterogeneity model. Information was generated for a marker locus linked to one of the disease-defining loci. Random-mating and assortative-mating samples were generated. Linkage analysis was then carried out by use of standard methods, under the assumptions of a single-locus disease trait and a random-mating population. Results were compared with those from analysis of a single-locus homogeneous trait in samples with the same levels of assortative mating as those considered for the two-locus traits. The results show that (1) introduction of assortative mating does not, in itself, markedly affect the estimate of the recombination fraction; (2) the power of the analysis, reflected in the LOD scores, is somewhat lower with assortative rather than random mating. Loss of power is greater with increasing levels of assortative mating; and (3) for a heterogeneous genetic disease, regardless of mating type, heterogeneity analysis permits more accurate estimate of the recombination fraction but may be of limited use in distinguishing which families belong to each homogeneous subset. These simulations also confirmed earlier observations that linkage to a disease "locus" can be detected even if the disease is incorrectly defined as a single-locus (homogeneous) trait, although the estimated recombination fraction will be significantly greater than the true recombination fraction between the linked disease-defining locus and the marker locus.     

Photosystem II Excitation Pressure and Development of Resistance to Photoinhibition (I. Light-Harvesting Complex II Abundance and Zeaxanthin Content in Chlorella vulgaris)

The basis of the increased resistance to photoinhibition upon growth at low temperature was investigated. Photosystem II (PSII) excitation pressure was estimated in vivo as 1 - qp (photochemical quenching). We established that Chlorella vulgaris exposed to either 5[deg]C/150 [mu]mol m-2 s-1 or 27[deg]C/2200 [mu]mol m-2 s-1 experienced a high PSII excitation pressure of 0.70 to 0.75. In contrast, Chlorella exposed to either 27[deg]C/150 [mu]mol m-2 s-1 or 5[deg]C/20 [mu]mol m-2 s-1 experienced a low PSII excitation pressure of 0.10 to 0.20. Chlorella grown under either regime at high PSII excitation pressure exhibited: (a) 3-fold higher light-saturated rates of O2 evolution; (b) the complete conversion of PSII[alpha] centers to PSII[beta] centers; (c) a 3-fold lower epoxidation state of the xanthophyll cycle intermediates; (d) a 2.4-fold higher ratio of chlorophyll a/b; and (e) a lower abundance of light-harvesting polypeptides than Chlorella grown at either regime at low PSII excitation pressure. In addition, cells grown at 5[deg]C/150 [mu]mol m-2 s-1 exhibited resistance to photoinhibition comparable to that of cells grown at 27[deg]C/2200 [mu]mol m-2 s-1 and were 3- to 4-fold more resistant to photoinhibition than cells grown at either regime at low excitation pressure. We conclude that increased resistance to photoinhibition upon growth at low temperature reflects photosynthetic adjustment to high excitation pressure, which results in an increased capacity for nonradiative dissipation of excess light through zeaxanthin coupled with a lower probability of light absorption due to reduced chlorophyll per cell and decreased abundance of light-harvesting polypeptides.     

Divergent evolution of a β/α-barrel subclass: Detection of numerous phosphate-binding sites by motif search

Study of the most conserved region in many β/α-barrels, the phosphate-binding site, revealed a sequence motif in a few β/α-barrels with known tertiary structure, namely glycolate oxidase (GOX), cytochrome b₂ (Cyb2), tryptophan synthase α subunit (TrpA), and the indoleglycerolphosphate synthase (TrpC). Database searches identified this motif in numerous other enzyme families: (1) IMP dehydrogenase (IMPDH) and GMP reductase (GuaC); (2) phosphoribosylformimino-5-aminoimidazol carboxamide ribotide isomerase (HisA) and the cyclase-producing D-erythro-imidazole-glycerolphosphate (HisF) of the histidine biosynthetic pathway; (3) dihydroorotate dehydrogenase (PyrD); (4) glutamate synthase (GltB); (5) ThiE and ThiG involved in the biosynthesis of thiamine as well as related proteins; (6) an uncharacterized open reading frame from Erwinia herbicola; and (7) a glycerol uptake operon antiterminator regulatory protein (GlpP). Secondary structure predictions of the different families mentioned above revealed an alternating order of β-strands and α-helices in agreement with a β/α-barrel-like topology. The putative phosphate-binding site is always found near the C-terminus of the enzymes, which are all at least about 200 amino acids long. This is compatible with its assumed location between strand 7 and helix 8. The identification of a significant motif in functionally diverse enzymes suggests a divergent evolution of at least a considerable fraction of β/α-barrels. In addition to the known accumulation of β/α-barrels in the tryptophan biosynthetic pathway, we observe clusters of these enzymes in histidine biosynthesis, purine metabolism, and apparently also in thiamine biosynthesis. The substrates are mostly heterocyclic compounds. Although the marginal sequence similarities do not allow a reconstruction of the barrel spreading, they support the idea of pathway evolution by gene duplication.     

Herpesvirus ateles DNA and Its Homology with Herpesvirus saimiri Nucleic Acid

Analysis of the structural organization of Herpesvirus ateles DNA shows that two types of viral DNA molecules are encapsidated in virions: (i) M-genomes, which contain 74% light sequences (L-DNA, 38% guanine plus cytosine) and 26% highly repetitive heavy sequences (H-DNA, 75% guanine plus cytosine), and (ii) defective H-genomes, which consist exclusively of repetitive H-DNA. The structure of M-genomes from H. ateles consists of an L-DNA region of about 70 x 10(6) daltons inserted between H-DNA termini of variable length. M-genomes with a shorter H-DNA region at one end of the molecule have a long stretch of H-DNA at the other end, resulting in a total molecular weight of 89.8 +/- 8.5 x 10(6). Thus it resembles the structure of M-genomes of H. saimiri. H-DNA of the two independent H. ateles isolates, strains 810 and 73, reveals different patterns after cleavage with restriction endonuclease Sma I. H-DNA of H. ateles 810 appears to consist of identical tandem repeat units with a molecular weight of 1,035,000; the H-DNA repeat unit of strain 73 is shorter (930,000 molecular weight). Corresponding DNA sequences of the two H. ateles strains (810 and 73) are completely homologous in cross-hybridizations. However, a discrete nucleotide sequence divergence between these virus strains is detected by measuring melting temperatures (T(m)) of DNA hybrid molecules. Some homology exists between H. ateles and H. saimiri DNA. Hybridization of L-DNA from H. ateles with L-DNA from H. saimiri shows about a 35% homology between the respective L-DNA sequences; the resulting heteroduplex molecules show a decrease of T(m) by 13.5 degrees C, corresponding to about a 9% mismatching in cross-hybridizing parts of L-regions. Very little homology is found between H-DNA of H. ateles and H. saimiri.     

Predicted Delays in the Activation of the Contractile System

The capacitance C'(e), presumed to be located across the walls of the transverse tubules of twitch fibers, was identified in earlier impedance measurements by virtue of having a resistance in series with it. When the voltage V(m) across the surface membrane is made to vary, the voltage V(c) across C'(e) will be delayed with respect to V(m), the extent of the delay depending on the location of the series resistance. Model 1 assumes that the resistivity of the lumen of the tubules is negligible; model 2 assumes that the series resistance arises entirely in the tubular lumen; model 3 assumes that the resistivity of the tubular lumen is small, but not negligible and that the bulk of the resistance arises in a structure directly in series with C'(e) and having a similar geometric distribution. If V(m) varies sinusoidally, the relative value of V(c(max)) will fall with increasingly higher powers of the frequency at the center of the fiber if model 2 is applicable, whereas models 1 and 3 predict that V(c(max)) will fall at high frequency only in proportion to the frequency everywhere in the cross-section of the fiber. Equations have been derived for the voltage change V(c) in response to a step change of V(m) and during an action potential. On the assumption that contraction is initiated when V(c) reaches mechanical threshold, the delay between the activation of myofibrils on the axis of the fiber and at the surface would amount to 2.6 msec in model 2 and 0.25 msec in model 3 for frog fibers of about 100 mum diameter during a twitch.