Restriction enzyme mapping of vaccinia virus DNA.

The cleavage sites for the restriction enzymes Bg/I, HindIII, KpnI, SalI, SmaI, and XhoI were located, from primary data, on the DNA isolated from the WR strain of vaccinia virus. Bg/I and SmaI divide the DNA into five segments which can be isolated by sucrose gradient centrifugation. These large segments provide a convenient means to group segments produced by other enzymes. The construction of physical maps was initiated by identifying the segments at each end of the DNA and then finding segments which were adjacent to these terminal sections. This was accomplished by isolating large shear fragments which contained the covalently linked termini of the DNA. Most of the data needed to derive the maps were obtained by isolating segments produce by one enzyme and then cleaving these individual segments with a second enzyme.     

The localization of glutathione peroxidase in the photoreceptor cells and the retinal pigment epithelial cells of Wistar and Royal College of Surgeons dystrophic rats

INTRODUCTION: If degenerating photoreceptor outer segments not phagocytized by RPE cells in the retina of Royal College Surgeons (RCS) rats were to undergo peroxidation, the distribution of glutathione peroxidase (GSH-PO) in the mitochondria or cytoplasm of the retina might be altered. We evaluated the immunocytochemical localization of GSH-PO to identify subcellular organelles in sections of the retinas of RCS rats. METHODS: Immunoblot analysis confirmed the presence of GSH-PO molecules in the retinas of RCS and Wistar rats aged 3 weeks. Sections were reacted with the F(ab) fragment of anti-rat alphaGSH-PO and then examined by laser scanning microscopy (LSM) and transmission electron microscopy (TEM). RESULTS: The size of the GSH-PO molecule in the retina was about 21 KD in the mitochondria and 23 KD in the cytosol in both strains of rats. LSM revealed fluorescent granules in the photoreceptor inner segments of the Wistar rats, and immunohistochemical TEM revealed GSH-PO in the mitochondria of their photoreceptor inner segments and retinal pigment epithelial (RPE) cells. In the RCS rats, the degenerating photoreceptor outer segments were clearly seen to be positive for anti-GSH-PO by conventional light microscopy (CLM). However, the photoreceptor inner segments of the RCS rats were negative for staining with anti-GSH-PO by LSM, and no GSH-PO could be detected in the mitochondria of the photoreceptor inner segments or RPE cells by immuno-TEM. CONCLUSION: Degeneration of the photoreceptor outer segments induced mitochondrial damage in the photoreceptor inner segments, and as a result GSH-PO shifted from the photoreceptor inner segments to the degenerating outer segments.     

Developmental and genotypic differences in the response of pea stem segments to auxin

The objective of this investigation was to examine the response to exogenous auxin (indole-3-acetic acid; IAA)of stem segments at two developmental stages. The standard auxin response of excised stem segments and intact plants consists of an initial growth response and a prolonged growth response. We found that this biphasic response does not occur in internodes at very early stages. Stem segments of light grown pea of various genotypes were cut when the fourth internode was at 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Length measurements of excised segments were made after 48 hours of incubation on buffer with or without auxin. An angular position transducer linked to a computerized data collection system provided high-resolution measurement of growth of stacks of segments incubated in buffer over 20 hours. Early-expansion segments of all genotypes deviated from the standard auxin response, while mid-expansion segments responded in a manner consistent with previous reports. Early-expansion segments of tall, light-grown plants were unique in showing an auxin-induced inhibition of growth. The auxin-induced inhibition correlated with high endogenous auxin content, as determined by HPLC and GC/MS, across genotypes and between early-expansion and mid-expansion segments of tall plants. Measurement of ethylene evolved from stem segments in response to auxin, and treatment of segments with the ethylene action inhibitor, norbornadiene, showed the inhibition to be mediated in part by heightened ethylene sensitivity. Growth of early-expansion segments of dwarf and severe dwarf plants was stimulated by exogenous auxin, but the growth rate increase was delayed compared to that in mid-expansion segments. This is the first time that such a growth response, termed the delayed growth response has been emonstrated. It is concluded that developmental stage and endogenous hormone content affect tissue response to exogenous auxin.     

Major role of positive selection in the evolution of conservative segments of Drosophila proteins

Slow evolution of conservative segments of coding and non-coding DNA is caused by the action of negative selection, which removes new mutations. However, the mode of selection that affects the few substitutions that do occur within such segments remains unclear. Here, we show that the fraction of allele replacements that were driven by positive selection, and the strength of this selection, is the highest within the conservative segments of Drosophila protein-coding genes. The McDonald-Kreitman test, applied to the data on variation in Drosophila melanogaster and in Drosophila simulans, indicates that within the most conservative protein segments, approximately 72 per cent (approx. 80%) of allele replacements were driven by positive selection, as opposed to only approximately 44 per cent (approx. 53%) at rapidly evolving segments. Data on multiple non-synonymous substitutions at a codon lead to the same conclusion and additionally indicate that positive selection driving allele replacements at conservative sites is the strongest, as it accelerates evolution by a factor of approximately 40, as opposed to a factor of approximately 5 at rapidly evolving sites. Thus, random drift plays only a minor role in the evolution of conservative DNA segments, and those relatively rare allele replacements that occur within such segments are mostly driven by substantial positive selection.     

Anti-lamprey retinal antibodies: immunohistochemistry on the retinas of several species of vertebrates

Summary Two monoclonal antibodies, H16 and B11, which were raised against lamprey retinal homogenate, were found to react with both short and long photoreceptor outer segments. On Western blotting of the retinal homogenate, both antibodies recognized a 40000 Da and a 80000 Da band. H16 antibody stained rod outer segments of all examined vertebrates, all cone outer segments of the turtle and chicken, and certain cone outer segments of the macaque. B11 antibody stained sub-mammalian rod outer segments and some mammalian cone outer segments, leaving all mammalian rod outer segments unstained. The epitope recognized by H16 antibody is considered to be located in a conserved or commonly inherited element of an outer segment-bound molecule, presumably rhodopsin. B11 antibody, on the other hand, seems to recognize a reactive group which has failed to be inherited by mammalian rod cells; why it recognizes all cone outer segments in the turtle and chicken and only a part of them in the cow, cat, and macaque, meanwhile ignoring all of them in the frog and fish, is subject to further study.A portion of this work is contained in a dissertation submitted by the first author, M.Y., in application for the Ph.D. degree at Yamagata University School of Medicine.     

The complete sequence of Drosophila alpha-spectrin: conservation of structural domains between alpha-spectrins and alpha-actinin

We report the complete sequence of Drosophila alpha-spectrin and show that it is similar to vertebrate nonerythroid spectrins. As in vertebrates, the alpha subunit consists of two large domains of repetitive sequence (segments 1-9 and 11-19) separated by a short nonrepetitive sequence (segment 10). The 106-residue repetitive segments are defined by a consensus sequence of 54 residues. Chicken alpha-spectrin (Wasenius, V.-M., M. Saraste, P. Salven, M. Eramaa, L. Holm, V.-P. Lehto. 1989. J. Cell Biol. 108:79-93) shares 50 of these consensus positions. Through comparison of spectrin and alpha-actinin sequences, we describe a second lineage of spectrin segments (20 and 21) that differs from the 106-residue segments by an 8-residue insertion and by lack of many of the consensus residues. We present a model of spectrin evolution in which the repetitive lineage of spectrin segments and the nonrepetitive lineage of segments found in spectrin and alpha-actinin arose by separate multiplication events.     

DNA inhibition and development of the pigment epithelium and receptors in rat retina]

In the retina of the guinea pig the outer segments of the receptor cells are damaged by cyclophosphamid, but the inner segments and the centriole are not altered. The disturbed membranes of the outer segments are incorporated by pigment epithelium. The progress of degeneration of outer segments corresponds with the increase of the number of Lysosomes in the pigmemt epithelium. The structure of mitochondria is disturbed by blocking the DNA. The unaltered mitochondria show a normal reaction of succinode hydrogenase.     

The characterization and comparison of amyloidogenic segments and non-amyloidogenic segments shed light on amyloid formation

Amyloid fibrillar aggregates of proteins or peptides are involved in the etiology of several neurodegenerative diseases and represent a major problem in healthcare. Short regions in the protein trigger this aggregation. It is important to understand the basis of such short regions aggregation and amyloidosis for therapeutic intervention. In this study, we describe specific physico-chemical properties of amyloidogenic segments and compare them with non-amyloidogenic segments. First, amyloidogenic segments are characterized by lower values for average net charge, electrostatic potential, solvent accessible surface area and B-factor when compared to the non-amyloidogenic segments of the same proteins. Second, they are enriched in hydrophobic residues and have a tendency to form hydrogen bonds. Thus, amyloidogenic segments have distinct physico-chemical properties that are different from those of non-amyloidogenic segments. Third, and quite unexpectedly, our dynamic simulation studies support the hypothesis that amyloidogenic segments have lower average flexibility than non-amyloidogenic segments. Furthermore, the presence of amyloidogenic segments in disordered proteins does not contradict the observation that amyloidogenic segments are less flexible.     

Homeotic gene function in the muscles of Drosophila larvae

The segmental musculature of Drosophila melanogaster larvae consists of 24-30 muscles per segment. Unique patterns of muscles are found in the three thoracic segments and the first and last abdominal segments; the remaining abdominal segments share the same pattern. Mutations in Ultrabithorax (Ubx) cause partial transformation of the muscle pattern of larval abdominal segments towards metathorax. The muscles of the thorax are not affected. In the first two abdominal segments the changes include the loss of at least 11 `abdominal' muscles and the gain of 11 `thoracic' muscles. Less extensive transformations are seen in more posterior abdominal segments. Anterobithorax, bithorax, postbithorax and bithoraxoid mutations also induce transformations of the larval musculature. Each allelic group affects a domain that is a subset of the entire Ubx domain but these domains are not restricted to compartments or segments and may extend through as many as five segments. In the muscles the segmental distribution of Ubx antigen correlates with the segments affected by Ubx mutations. The different domains of Ubx in mesoderm and ectoderm argue that the segmental diversity of the muscle pattern is not simply induced by the overlying epidermis and that Ubx function in the mesoderm is required for the correct development of abdominal segments.     

Kinetic analysis of root growth and georeaction

The kinetics of growth and georeaction of horizontal or vertical maize (cv. ORLA 264) apical root segments were analysed. Elongation and curvature data were fitted to a mathematical function and the effect of both light and decapitation reported. Elongation of horizontal segments was found to be more sensitive to light than to decapitation. A light treatment or the decapitation presented several effects on the shape of the growth curves. Growth of vertical segments was affected much more by decapitation than by light treatment. The shape of the curves was clearly different for decapitated and intact segments.
Curvature is affected both by light and decapitation but the shape of the bending curves is modified principally by decapitation.
Under the present conditions used, growth and georeaction of root segments do not seem to be strictly correlated.     

Rapid Initiation of Thymidine Incorporation into Deoxyribonucleic Acid in Vegetative Tobacco Stem Segments Treated with Indole-3-acetic Acid

The short term effect of 11.4 mum indoleacetic acid on the incorporation of (methyl-(3)H)thymidine into DNA in vegetative tobacco (Nicotiana tabacum cv. Wis. 38) stem segments has been investigated. In segments that are defoliated, inverted, and kept in the dark for 7 hours, indoleacetic acid very rapidly (about 60 minutes) and strikingly initiates thymidine incorporation into DNA. The time required before enough indoleacetic acid (2.8 mum) to enhance thymidine incorporation moves into a segment has been found to be about 35 minutes. The initiation response time for segment tissue that already contains 2.8 mum indoleacetic acid should be no more than about 25 minutes. The rate of labeled thymidine incorporation into DNA is affected by physiological treatments of segments. Moving segments from the light into the dark or defoliating segments or inverting defoliated segments decreases the rate of thymidine incorporation. For segments given all three treatments, indoleacetic acid restores the rate of thymidine incorporation as compared to controls. Darkness, or defoliation or inversion of segments, therefore, may decrease thymidine incorporation into DNA by effecting reduced auxin levels in stem segments.     

Retention function of all segments of IUD usage: a compound distribution.

The distribution of the all segments of IUD use consists of two components: the distribution of the length of each segment and the distribution of the number of segments. In this article, a compound distribution of all segments of IUD, developed by the authors is described. The distribution assumes that each segment of IUD use is a mixed exponential distribution and the number of segments experienced by women is a truncated Poisson distribution. This distribution is useful in estimating the demographic impact of an IUD and IUD program.     

The segments of influenza viral mRNA.

Influenza viral mRNA, i.e., complementary RNA (cRNA), isolated from infected cells , was resolved into six different species by electrophoresis in 2.1% acrylamide gels containing 6 M urea. The cRNA''s were grouped into three size classes: L (large), M (medium-size), and S (small). Similarly, when gels were sliced for analysis, the virion RNA (vRNA) also distributed into six peaks because the three largest vRNA segments were closely spaced and were resolved only when the gels were autoradiographed or stained. Because of their attached polyadenylic acid [poly(A)]sequences, the cRNA segments migrated more slowly than did the corresponding vRNA segments during gel electrophoresis. After removal of the poly(A) by RNase H, the cRNA and vRNA segments comigrated, indicating that they were approximately the same size. One of the cRNA segments, S2, was shown by annealing to contain the genetic information in the vRNA segment with which it comigrated, strongly suggesting that each cRNA segment was transcribed from the vRNA segment of the same size. In contrast to the vRNA segments, which when isolated from virions were present in approximately 1:1 molar ratios, the segments of the isolated cRNA were present in unequal amounts, with the segments M2 and S2 predominating, suggesting that different amounts of the cRNA segments were synthesized in the infected cell. The predominant cRNA segments, M2 and S2, and also the S1 segment, were active as mRNA''s in wheat germ extracts. The M2 cRNA was the mRNA for the nucleocapsid protein; S1 for the membrane protein; and S2 for the nonstructural protein NS1.     

Segmentation of moving images by the human visual system

 New segments appearing in an image sequence or spontaneously accelerated segments are band limited by the visual system due to a nonperfect tracking of these segments by eye movements. In spite of this band limitation and acceleration of segments, a coarse segmentation (initial segmentation phase) can be performed by the visual system. This is interesting for the development of purely automatic segmentation algorithms for multimedia applications. In this paper the segmentation of the visual system is modelled and used in an automatic coarse initial segmentation. A suitable model for motion processing based on a spectral representation is presented and applied to the segmentation of synthetic and real image sequences with band limited and accelerated moving foreground and background segments. Received: 1 August 1995/Accepted in revised form: 25 February 1997     

The structure of A segments from glycerinated skeletal muscle.

The A band of skeletal muscle consists of an array of thick myosin-containing filaments along with non-myosin proteins such as C protein and M line protein. In order to study the arrangement of the myosin and non-myosin components, A segments which are aggregations of thick filaments held together at the M line were prepared from glycerinated chicken pectoral and rabbit psoas muscles and examined by electron microscopy. Details of the preparative technique and comparison of the morphologies of A segments and I segments are provided. The A segments from chicken pectoral muscle exhibited 11 to 12 stripes in each half lateral to the bare zone. Several less distinct bands as well as subdivisions of the individual stripes were also observed. The periodicity of the major stripes in the A segments was 424 +/- 10 A. The A segments prepared from rabbit psoas muscle had a periodicity of 432 +/- 13 A, but in contrast with chicken A segments, fewer rabbit A segments showed this periodicity. We conclude that A segments can be separated from glycerinated chicken and rabbit skeletal muscles and compare our results with those of others who prepared A segments from frog and rabbit skeletal muscles in the absence of glycerol.     

Effects of Cytokinins on the Growth of Primary Roots of Zea mays

Intact primary roots of Zea mays seedlings, apical 6-mm segmentsisolated from the intact primary roots and 5-mm detipped segments,prepared from the 6-mm apical segments by removal of the apical1-mm meristematic region, were incubated in potassium-phosphatebuffer that contained various concentrations of kinetin, 6-benzylaminopurine(BAP) or zeatin. These cytokinins inhibited the growth of intactprimary roots but they promoted the growth of both tipped anddetipped apical segments. In other words, they promoted thegrowth of root segments irrespective of the presence or absenceof apical meristematic regions. Detipped segments were stoodvertically, with their apical or basal cutends in contact withan agar plate that contained the abovementioned buffer and variousconcentrations of kinetin, BAP or zeatin so that cytokininswere supplied either from the apical or basal cut-ends. Cytokininssupplied from the top promoted the growth of the segments, whilethose supplied from the base did not. These results indicate that the response of roots to the exogenouslyapplied cytokinins is not influenced by the presence of theroot meristem but is significantly affected by the way in whichcytokinins are supplied. (Received October 17, 1995; Accepted August 28, 1995)     

Differential distribution of pigment-protein complexes in the Thylakoid membranes of Synechocystis 6803

Thylakoids in Synechocystis 6803, though apparently uniform in appearance in ultrastructure, were found to consist of segments which were functionally dissimilar and had distinct proteomes. These thylakoid segments can be isolated from Synechocystis 6803 by successive ultracentrifugation of cell free extracts at 40,000×g (40?k segments), 90,000×g (90?k segments) and 150,000×g (150?k segments). Electron microscopy showed differences in their appearance. 40?k segments looked feathery and fluffy, whereas the 90?k and 150?k thylakoid membrane segments appeared tiny and less fluffy. The absorption spectra showed heterogeneous distribution of pigment-protein complexes in the three types of segments. The photochemical activities of Photosystem I (PSI) and Photosystem II (PSII) showed unequal distributions in 40?k, 90?k and 150?k segments which were substantiated with low temperature fluorescence measurements. The ratio of PSII/PSI fluorescence emission at 77?K (λ(ex)?=?435?nm) was highest in 150?k segments indicating higher PSII per unit PSI in these segments. The chlorophyll fluorescence lifetimes in the membranes, determined with a time-correlated single-photon counting technique, could be resolved in three components: τ(1) (=)?<40?ps, τ(2) (=)?425-900?ps and τ(3) (=)?2.4-3.2?ns. The percentage contribution of the fastest component (τ(1)) decreased in the order 40?k?>?90?k?>?150?k segments whereas that of the other two components showed a reversed trend. These studies indicated differential distribution of pigment-protein complexes in the three membrane segments suggesting heterogeneity in the thylakoids of Synechocystis 6803.     

Developmental changes in the gibberellin-induced growth response in stem segments of light-grown pea genotypes

The effects of GA on stem elongation were studied using segments from one tall and three dwarf light-grown pea genotypes varying in endogenous hormone content. Stem segments were cut at two distinct ages: when the fourth internode was at about 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Light microscopy and flow cytometry were used to demonstrate that GA does not induce cell division in excised pea stem segments. The growth studied here was strictly elongation. Measurement of final segment length after 48 hours and high resolution measurement of growth kinetics over 20 hours using an angular position transducer were done on segments treated with hormone solutions. Our data indicate that the action of GA on stem elongation can be classified into two distinct modes. The first, apparent in early-expansion stem segments, shows distinct growth kinetics and is independent of the endogenous IAA concentration of the segments. Quantitation of IAA by GC/MS in early-expansion segments of wild type pea incubated with gibberellin shows that an increase in IAA concentration is part of the GA response in such segments. The second mode of GA action is evinced in mid-expansion segments. Whereas there is no short term (<20 h) response to GA alone (as determined by growth kinetics), there is a long term (48 h) response whose magnitude decreases across the genotypes with decreasing endogenous hormone content. Growth responses indicate that in mid-expansion segments exogenous GA acts by enhancing IAA action but appears to be unable to augment endogenous IAA content. Contradictory reports of the response of excised stem segments to GA can be reconciled when tissue genotype and developmental stage are considered.     

Encapsulation, cold storage, and growth of Hibiscus moscheutos nodal segments

Nodal segments of Hibiscus moscheutos (hardy hibiscus) were excised from proliferating axillary shoot cultures and encapsulated in high density sodium alginate hardened by 50 mM CaCl₂. Nodal segments 4 mm long grew as well as and were easier to encapsulate than 8 mm long nodal segments. Although nodal segments grew regardless of the concentration of sodium alginate, 2.75% was determined to produce the highest quality encapsulated nodal segments beads (sufficient alginate coating and ease of use) because of the viscosity produced by the 2.75% sodium alginate solution. When encapsulated segments were stored at 5°C they did not grow in light or darkness. During the first month on fresh proliferation medium under normal incubation conditions following 5°C storage in the dark for up to 24 weeks, root number and root and shoot elongation were inhibited linearly as storage time increased. All encapsulated nodal segments survived 24 weeks of 5°C storage in two separate experiments. In fact, 80% of encapsulated hardy hibiscus nodal segments survived refrigerated storage for 1½ years (78 weeks) and after 3 months on proliferation medium, the nodal segments produced nearly the same length axillary shoots with the same number of axillary nodes per shoot as compared to encapsulated segments either not stored at 5°C or stored for 24 weeks at 5°C. Growth from encapsulated and cold-stored ‘Lord Baltimore’ nodal segments was more vigorous than from ‘Southern Belle’ nodal segments.     

DNA segments mapped by reciprocal use of microsatellite primers between mouse and rat

Rat microsatellite primers were used for detection of homologous DNA segments in the mouse species (Mus laboratorius, Mus musculus musculus, and Mus spretus). Twenty five (16.3%) of 153 rat primer pairs amplified specific DNA segments, when genomic DNA of mice was used as a template in the polymerase chain reaction (PCR). Size variation among inbred strains of mice was found for 13 DNA segments (8.5%). Eight out of the 13 polymorphic DNA segments were mapped to a particular chromosome with two sets of recombinant inbred strains, AKXL or BXD. Similarly, mouse microsatellite primers were used for detection of homologous DNA segments in rats (Rattus norvegicus). Twenty (12.0%) of 166 primer pairs amplified specific DNA segments from rat genome. Size variation among inbred strains of rats was found for seven DNA segments (4.2%). Eleven of these 20 DNA segments were mapped with a rat x mouse somatic cell hybrid clone panel and/or linkage analysis by use of backcross progeny. Our results suggest that the mapped DNA segments are really homologs between mouse and rat. These polymorphic DNA segments are useful genetic markers.