Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.     

Inhibition of IL 2-dependent proliferation of rat T lymphoblasts by the monoclonal antibody ART62 which reacts with MHC class 1 antigens

During the course of studies designed to obtain monoclonal antibodies (mAb) that recognize the rat interleukin 2 receptor, a mouse IgG1 mAb (ART62) was identified which inhibits the interleukin 2 (IL 2)-dependent proliferation of rat T lymphoblasts without affecting the binding of IL 2 to such cells. In order to characterize the cell surface components that react with the mAb ART62, T lymphoblasts were surface-labeled with 125I, and the radioactive molecules were immunoprecipitated by the antibody analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The mAb ART62 precipitated two major components of 48,000 m.w. and 12,000 m.w., respectively, which were different from those which react with the anti-IL 2-receptor antibody ART18, a molecule of 50,000 to 55,000 m.w. Sequential immunoprecipitation studies revealed that the mAb ART62 reacts with the MHC class 1 antigen that reacts with the classical anti-rat MHC class 1 mAb OX18, and vice versa. In contrast to the mAb ART62, OX18 that does not affect and several other mAbs known to inhibit the rat MLR failed to inhibit IL 2-dependent proliferation of rat T lymphoblasts. In contrast to the anti-IL 2 receptor antibody ART18, ART62 effectively inhibited IL 2-driven proliferation even when added to cells already committed to proliferate by IL 2-IL 2 receptor interaction. These data raise the possibility that MHC class 1 antigens could be involved in the chain of reactions mediating the signals required for cell proliferation.     

Isolation, characterization and chromosomal mapping of the mouse tyrosine aminotransferase gene

The tyrosine aminotransferase (TAT) gene is expressed in a tissue and developmental-specific manner. In addition, this gene is regulated by glucocorticoid and polypeptide hormones and its expression is affected when a regulatory region near the albino locus of the mouse is deleted. In order to allow studies of the molecular effects of these deletion mutations we have isolated and characterized the mouse TAT gene. The gene is 9.2 x 10(3) bases in length and consists of 12 exons which give rise to a 2.3 x 10(3) base long messenger RNA. The DNA sequence at the 5' end of the gene was determined and compared with the corresponding sequence of the rat tyrosine aminotransferase gene. The sequence comparison showed extensive homology over the entire region sequenced. In addition, DNA: DNA heteroduplex studies between the mouse and rat tyrosine aminotransferase genes revealed that this homology extends over the entire gene and its flanking sequences. The mouse tyrosine aminotransferase gene has been mapped distal to the serum esterase-1 locus on mouse chromosome 8, using a restriction fragment length polymorphism between two mouse species. Since the albino deletions are located on mouse chromosome 7, the assignment of the TAT gene to chromosome 8 suggests that a regulatory factor(s) affecting TAT gene expression acts in trans.     

Disruption of microtubules in living cells and cell models by high affinity antibodies to beta-tubulin.

Polyclonal antibodies with high affinity for beta-tubulin were found to disrupt cytoplasmic microtubules efficiently after microinjection into tissue culture cells. The degree of microtubular fragmentation was directly proportional to the amount of the injected antibody. At molar ratios of 1 antibody per 100 tubulin dimers, most microtubules were disrupted within 90 min after injection. In contrast, the time course of disintegration was relatively independent of the antibody concentration. Within the range of 1 antibody per 10(2)-10(4) tubulin dimers, the maximal values for microtubular disintegration were reached approximately 1-1.5 h after injection. Mitotic microtubules were found to be resistant to all antibody concentrations used. In living cells, microtubules recovered within a few hours after antibody-induced decay. The time course of recovery, like the extent of disintegration, was a function of the antibody concentration. The antibody acted also on microtubules in detergent-extracted cell models and on microtubules polymerised in vitro. When added to microtubular protein, the bivalent antibody as well as its Fab fragments prevented polymerisation. The data suggest that these antibodies disrupt microtubules because their affinity to tubulin is at least 100 times higher than the affinities found for tubulin:tubulin interaction. Fragmented microtubules are probably unstable and decompose into smaller units.     

Animal community structure as a function of stream size

The species-area relationship of the island biogeography theory was calculated for macroinvertebrates in 22 coastal, adjacent streams. A z-value of 0.19 was obtained. The low z-value was probably a consequence of the short distances between streams as well as high dispersal rates. In addition, a cluster analysis based on the dissimilarity of species assemblages showed that stream size was of prime importance in categorizing the streams. To a smaller extent water quality affected the community structure in the streams.     

Mapping of cysteine genes on the chromosome of Pseudomonas aeruginosa PAO

Summary Three loci coding for different steps in the pathway of cysteine biosynthesis have been mapped by R68.45-mediated coconjugation analysis. The cysteine auxotrophic mutants could be subdivided into sulfite and sulfide-requiring mutants. Sulfide-requiring mutants (cysIV group) were localized at a single position between pyrF and pur-67, while sulfite-requiring mutants (cysI and cysII) mapped at two different regions. The cysI group was also localized between pyrF and pur-67, although more distal to pyrF than the cysIV group. This group included the cys-54 marker, which has been mapped previously. The second group of sulfite-requiring mutants, designated as cysII, was cotransducible with hisI and localized at the end of the PAO chromosomal map. This location was also confirmed for the marker cys-59.The marker cys-59 (which was cotransducible with his1) was cotransferred by R68.45-mediated conjugations with both the late marker pur-67 and the early marker ilv-226. As the late marker hisI was positioned at about 60–65 min (Herrmann and Günther, in press) the length of the PAO chromosome was estimated to be about 70 min.     

Use of monoclonal antibodies as a diagnostic tool in human leukemia. I. Acute myeloid leukemia and acute phase of chronic myeloid leukemia

Various monoclonal antibodies (mAbs) detecting certain different epitopes on myeloid cells (VIMD5, D5 D6, OKM1, Leu-M3, VIEG4, OKIa 1) have been used in combination with conventional markers (antihuman myeloid hetero-antiserum, FcIgG-receptors, C3d-receptors) to further define the phenotypic heterogeneity of myeloid leukemia. Subsequent leukemic samples from previously untreated patients with acute myeloid leukemia (AML) (51 adults, 24 children) and from nine adult patients in the acute phase of chronic myeloid leukemia (CML-BC) were studied. It was possible to demonstrate quantitative differences in the expression of antigens on the various leukemia subtypes which could be exploited for diagnosis. Furthermore our results revealed that there is a very close correlation between the different surface phenotypes and the types morphologically assessed according to FAB-criteria.     

Determination of Melting Sequences in DNA and DNA-Protein Complexes by Difference Spectra

A graphical formula is presented for determining the base ratio of melted DNA. By use of this formula, the composition of sequences which melt in different portions of the melting curves of Clostridium DNA, Escherichia coli DNA, and mouse DNA were determined. As the DNA melts, the per cent of adenine and thymine (AT) in the melted sequences decreases linearly with temperature. The average composition of sequences which melt in a given part of the melting curve is proportional to the base ratio of the DNA. The concentration and average composition of sequences were determined for three parts of the melting curves of the DNA samples, and a frequency distribution curve was constructed. The curve is symmetrical and has a maximum at about 56% AT. The distribution of GC-rich sequences on the E. coli chromosome was estimated by shearing, partially melting, and fractionating the DNA on hydroxylapatite. GC-rich sequences appear to occur every thousand base pairs, and have a maximum length of about 180 base pairs. The graphical formula was applied to the determination of the composition of sequences which melt in different parts of the melting curve of chromatin. Throughout the melting curve, the composition of the melting sequences is about 60% AT, which appears to suggest that relatively long sequences are melting simultaneously. Their melting temperature may be a function of the composition of the protein on different parts of the DNA. The problem of light scattering in DNA-protein and DNA was also investigated. A formula is presented which corrects for light scattering by relating the intensity of the scattered light to the rate of change of absorbance of DNA with wavelength.