DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r

Summary The complete nucleotide sequences of therecA genes fromEscherichia coli B/r,Shigella flexneri, Erwinia carotovora andProteus vulgaris were determined. The DNA sequence of the coding region of theE. coli B/r gene contained a single nucleotide change compared with theE. coli K12 gene sequence whereas theS. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to theE. coli K12 RecA protein. The DNA sequences of the recA genes fromE. carotovora andP. vulgaris were 80% and 74% homologous, respectively, to theE. coli K12 gene. The predicted amino acid sequences of theE. carotovora andP. vulgaris RecA proteins were 91% and 85% identical respectively, to that ofE. coli K12. The RecA proteins from bothP. vulgaris andE. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.     

Tissue to tissue symplastic communication in the shoots of etiolated corn seedlings

Carboxyfluorescein, a symplastic probe, was applied to the cut mesocotyl base or coleoptile apex of etiolated Zea mays cv. Silver Queen seedlings and its transport measured and tissue distribution determined. Long-distance longitudinal symplastic transport of the carboxyfluorescein was mainly in the vascular stele. It moved laterally from the mesocotyl stele to the mesocotyl cortex but the presence of a weak barrier limited the movement. A partial symplastic barrier was also present near the coleoptile-mesocotyl node.     

The septate junction limits mobility of lipophilic markers in plasma membranes ofHydra vulgaris (attenuata)

Summary Fluorescent lipophilic probes were used to study the role of septate junctions in maintaining distinct apical and basolateral domains of plasma membranes in epithelial cells of hydra. In short-term experiments, a 16-carbon chain aminofluorescein probe (AFC₁₆) was localized to the apical plasma membranes of ectodermal and endodermal epithelial cells when presented in the culture medium or injected into the gastric lumen, but did not demarcate basolateral membranes. In longer term experiments, basolateral membranes were stained and the staining was independent of temperature conditions. A dual 18-carbon chain indocarbocyanine probe (DiIC₁₈) gradually diffused across the septate junction to label basolateral membranes at room temperature, but not at 4°C. DiIC₁₈ also filled and stained certain mounted nematocytes. The results indicate that in hydra, lipophilic probes may be limited in mobility within the membrane plane by the septate junctions in a manner similar to vertebrate tight junctions, and that apical membranes of mature nematocytes are differentially permeable.     

Chromosomal domains of chimpanzee are diverged from human as revealed by in situ hybridization using human genomic probe

Utilization of repetitive DNA probes to assess the taxonomic affinity between related species has become the most powerful tool in evolutionary biology today. Consequently, tremendous strides have recently been made towards establishing the phylogenetic relationship of humans with chimpanzee. We employed human genomic proe (P5080 B.5) to identify the degree of divergence of chimpanzee genome from humans. A small protion of structurally distinct genomic areas in chimpanzee could be identified by fluorescencein situ hybridization (FISH) technique when compared to human DNA. The genomic divergence is confined mainly to the chromosomal ends in chimpanzee and may be an important phylogenetic characteristic in human evolution.     

Effect of high KCl concentrations on membrane-localized metastable proton buffering domains in thylakoids

Recent work showed that chloroplast thylakoid membranes stored in 100 mM KCl-containing media have delocalized energy coupling consistent with a rapid equilibration of the proton gradient between the proton-producing redox steps and the lumen bulk phase (Beard and Dilley 1986). Thylakoids stored in low salt media showed localized energy coupling. A related thylakoid membrane property is the occurrence of sequestered, metastable, acidic domains, associated with pK _a 7.5 amine groups. For low salt-stored membranes the domain protons appear to be in the direct (localized) diffusion pathway of protons involved in energizing ATP formation, whereas in thylakoids stored in high KCl, domain protons equilibrated with the lumen during the development of the ATP energization threshold (Theg et al. 1988). This work tested whether the 100 mM KCl storage treatment did or did not cause the dissipation of the metastable acidic domain protons in the dark, storage period. By three criteria, it was found that the 100 mM KCl storage treatment had only a slight tendency to dissipate the acidic domain protons into alkaline media under dark conditions. Storage in KCl does not cause the dissipation of the acidic domains in the dark, but allows domain protons to equilibrate with the lumen after the redox system begins turning over, but before the ATP energization threshold pH is reached. These results must be considered in models of how the thylakoid structure can accommodate metastable acidic domains and how such domain protons diffuse to the CF₀-CF₁ complexes in energy coupling.Abbreviations PSII photosystem 2 - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2(N-morpholine) ethanesulfonic acid - Taps N-tris (hydroxymethyl)-methyl-3-amino-propanesulfonic acid - EPPS N-(2-hydroxyethyl) piperazine-N-3-propanesulfonic acid - gram D gramicidin D This research supported in part by grants from the USDA and NSF.     

Fatty-acid spin probe interactions with erythrocyte ghosts and liposomes prepared from erythrocyte ghosts

Summary A model for the binding of 5-nitroxide stearate, I(12,3). to human erythrocyte ghosts was developed by comparing spin probe interactions with ghosts and liposomes prepared from ghosts. At low probe/lipid (P/L<1/2500), I(12,3) binds to a similar class of high-affinity, noninteracting sites in both ghosts and liposomes, indicating that lipid moieties are responsible for probe uptake. Saturation occurs in both systems with increasing P/L, and, at higher loading (e.g., P/L=1/360 for ghosts and liposomes), the probe inserts itself at initially dilute sites to form a class of low-affinity sites consisting of clusters of variable size. At still higher P/L ranges (>1/100), much increased probe uptake was observed in ghosts than in liposomes, which was attributed to another class of low-affinity sites, representing nonspecific interactions of I(12,3) with membrane proteins. The nature of the spectral components and ultrafiltration experiments with ghosts labeled at high P/L indicate that both dilute and clustered I(12,3) are due to membrane-incorporated probe.     

Dynamin-mediated Internalization of Caveolae

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.     

钙/钙调蛋白依赖性丝氨酸蛋白激酶的结构和功能

钙/钙调蛋白依赖性丝氨酸蛋白激酶(calcium/calmodulin-dependent serine protein kinase, CASK)属于膜相关鸟苷酸激酶（membrane associated guanylate kinase, MAGUK）家族.CASK具有多个不同蛋白质结合结构域，在细胞膜的特定区域，与其他蛋白质形成多种蛋白质复合体，参与组成细胞骨架.它通过衔接细胞外信号蛋白和细胞内骨架蛋白，协助功能蛋白质的转运和定位，以及细胞内的信号传递.此外CASK还可以进入细胞核影响基因转录调控，以及作用在神经突触膜上参与神经递质的释放.     

Modular organization of inteins and C-terminal autocatalytic domains.

Analysis of the conserved sequence features of inteins (protein "introns") reveals that they are composed of three distinct modular domains. The N-terminal (N) and C-terminal (C) domains are predicted to perform different parts of the autocatalytic protein splicing reaction. An optional endonuclease domain (EN) is shown to correspond to different types of homing endonucleases in different inteins. The N domain contains motifs predicted to catalyze the first steps of protein splicing, leading to the cleavage of the intein N terminus from its protein host. Intein N domain motifs are also found in C-terminal autocatalytic domains (CADs) present in hedgehog and other protein families. Specific residues in the N domain of intein and CADs are proposed to form a charge relay system involved in cleaving their N-termini. The intein C domain is apparently unique to inteins and contains motifs that catalyze the final protein splicing steps: ligation of the intein flanks and cleavage of its C terminus to release the free intein and spliced host protein. All intein EN domains known thus far have dodecapeptide (DOD, LAGLI-DADG) type homing endonuclease motifs. This work identifies an EN domain with an HNH homing-endonuclease motif and two new small inteins with no EN domains. One of these small inteins might be inactive or a "pseudo intein." The results suggest a modular architecture for inteins, clarify their origin and relationship to other protein families, and extend recent experimental findings on the functional roles of intein N, C, and EN motifs.