Fractionation, isolation and characterization of nonhistone chromosomal protein from calf thymus.

1) A method is described for the separation and fractionation of nonhistone chromosomal proteins from salt-urea dissociated calf thymus chromatin. After precipitating DNA in the dissociated chromatin solution with LaCl3, the chromosomal proteins in the supernatant were fractionated by SP-Sephadex C-25 column chromatography using a combination of NaCl stepwise and linear gradient elutions. Much care was taken to prevent proteolytic degradation of the chromosomal proteins during the preparation. 2) Among the protein fractions separated by this chromatography, twenty subfractions were found to be homogeneous on SDS-polyacrylamide gel electrophoresis. These purified proteins account for about 18% of the whole chromosomal protein. Eleven subfractions of these purified nonhistone proteins had ratios of acidic to basic amino acids above 1.0 and the nine remaining subfractions had ratios below 1.0, corresponding to nonhistone proteins of basic character. 3) The molecular weights of the purified nonhistone proteins ranged from 7,400 to 19,000.     

Rapid turnover of non-histone chromosomal proteins in skeletal muscle

Incorporation of ³H-leucine into histones and non-histone chromosomal proteins was investigated in liver, a tissue in which proteins generally turn over rapidly, and in muscle, a tissue in which proteins turn over slowly. Incorporation into histones was low in both tissues. Incorporation into non-histone chromosomal proteins which, in liver, proceeded at about the same rate as into soluble cytoplasmic proteins was, in muscle, considerably more rapid than into any other cytoplasmic or nuclear protein fraction investigated. The significance of the relatively high incorporation rate into the non-histone chromosomal proteins in muscle is not known. However, autoradiographic experiments suggest that in muscle all nuclei display a high rate of incorporation into these proteins, and gel electrophoretic experiments indicate that a high rate of turnover is characteristic of many of the proteins comprising this fraction.     

Aperiplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins

A search was performed for a periplasmic molecular chaperone which may assist outer membrane proteins of Escherichia coli on their way from the cytoplasmic to the outer membrane. Proteins of the periplasmic space were fractionated on an affinity column with sepharose-bound outer membrane porin OmpF. A 17kDa polypeptide was the predominant protein retained by this column. The corresponding gene was found in a gene bank; it encodes the periplasmic protein Skp. The protein was isolated and it could be demonstrated that it bound outer membrane proteins, following SDS-PAGE, with high selectivity. Among these were OmpA, OmpC, OmpF and the maltoporin LamB. The chromosomal skp gene was inactivated by a deletion causing removal of most of the signal peptide plus 107 residues of the 141-residue mature protein. The mutant was viable but possessed much-reduced concentrations of outer membrane proteins. This defect was fully restored by a plasmid-borne skp gene which may serve as a periplasmic chaperone.     

Characterization of the F plasmid TraJ protein synthesized in F' and Hfr strains of Escherichia coli K-12

Using purified F plasmid TraJ protein (Cuozzo, M., Silverman, P., and Minkley, E. (1984) J. Biol. Chem. 259, 6659-6666), we prepared rabbit anti-TraJ protein antibodies to analyze for the first time the TraJ protein as it is synthesized in normal F' and Hfr conjugal donor strains. Using affinity-purified antibody, we identified the protein on immuno-overlay blots of whole cell proteins separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In contrast to the TraJ protein synthesized in large quantity by heat-induced lambda (traJ) lysogens, the TraJ protein synthesized in normal donor cells was soluble, even after sedimentation at 100,000 X g. The soluble protein was found with the cytoplasmic fraction after separation of cytoplasmic and periplasmic proteins. Velocity sedimentation analysis indicated an S20,w of 3.5 for the single molecular species composed of or including all the TraJ polypeptide in crude extracts. Quantitative analyses showed that conjugal donor strains normally contain 2000-4000 TraJ monomers/cell. However, that level depended on other plasmid and chromosomal genes.     

Some observations on protein metabolism in chromosomes of non-dividing cells

1. The metabolism of chromosomal proteins has been studied in the pancreas, liver, and kidney of adult mice (a) by measuring the rates of glycine-N¹⁵ incorporation into histones and residual chromosome fractions, and (b) by measuring the extent to which N¹⁵, once incorporated into chromosomal proteins, is retained. 2. The uptake of isotopic nitrogen by these nuclear constituents was compared with that of protein fractions prepared from the cytoplasm by differential centrifugation in sucrose solutions. One such fraction, which comprises the bulk of the ribosenucleoprotein of the cell sediments as a pellet on high speed centrifugation. The supernatant remaining after this centrifugation is a fraction which, in the pancreas, is rich in the secretory enzymes synthesized by the cell. 3. A comparison of the rates of glycine-N¹⁵ uptake shows that cytoplasmic ribonucleoprotein is the most active of the protein fractions analyzed. In the pancreas it meets the conditions required of a precursor for the secretory enzymes of the supernate. 4. In all tissues considered the rates of glycine-N¹⁵ uptake into histone and residual chromosome fractions are lower, that for histone being the lowest of any of the protein components considered and that for residual protein approximating the over-all rate for cytoplasmic protein. 5. The effects of feeding and fasting upon glycine-N¹⁵ incorporation have been studied. In the pancreas, feeding causes a sharp increase in N¹⁵ uptake by the mixed tissue proteins and by the nucleoprotein and supernatant protein of the cytoplasm. There is a parallel increase in N¹⁵ uptake by the chromosomal constituents—histone and residual protein. 6. A parallelism between N¹⁵ uptake in cytoplasmic and chromosomal proteins is also observed in the liver and kidney when over-all protein metabolism is altered by feeding and fasting. 7. The responsiveness of the histones and residual proteins to changes in the environment has also been demonstrated in N¹⁵ retention experiments. The loss of isotope once incorporated into chromosomal proteins is much more rapid in fed than in fasted animals.     

Actin-Based Gelation of Cytoplasmic Extracts from Brain Tissue

Incubation of rat brain cytoplasmic extracts under the conditions described in this paper results in the formation of three-dimensional gels. Ultrastructurally, these gels correspond to complex supramolecular structures formed by single microfilaments and by microfilament bundles. Analysis of protein composition indicates that cytoplasmic gels are composed of actin and several associated proteins. Among the latter class of components, we have identified polypeptides with molecular weights of 55,000 (55K), 140,000 (140K), and a set of two or three polypeptides with molecular weights in the range of 235,000-245,000 (235-245K). The 55K and 140K components do not seem to correspond to any previously identified actin-associated proteins, while the 235-245K polypeptides may correspond to the protein known as fodrin.     

Proteins of Friend leukemia cells. Comparison of hemoglobin-synthesizing and noninduced populations.

Proteins of Friend leukemia cells induced to form large amounts of hemoglobin by dimethylsulfoxide treatment were compared with proteins from noninduced cells by high resolution two-dimensional polyacrylamide gel electrophoresis. Approximately 98% of more than 500 proteins separated by this technique were qualitatively and quantitatively the same in both cell populations. Changes representing more than 50% of the control cell amount were detected in six non-histone chromosomal proteins, two nucleoplasmic proteins, and three cytoplasmic proteins. It is concluded that dimethylsulfoxide induces an extremely specific pattern of erythroid differentiation in these cells, which should be susceptible to detailed analysis. Comparison of protein patterns from Friend leukemia cells and HeLa cells revealed electrophoretic identity of approximately 20% of cytoplasmic proteins and 50% of non-histone chromosomal proteins.     

Nucleotide sequences and expression in Escherichia coli of the in-phase overlapping Pseudomonas aeruginosa plcR genes.

The translation products of chromosomal DNAs of Pseudomonas aeruginosa encoding phospholipase C (heat-labile hemolysin) have been examined in T7 promoter plasmid vectors and expressed in Escherichia coli cells. A plasmid carrying a 4.7-kilobase (kb) DNA fragment was found to encode the 80-kilodalton (kDa) phospholipase C as well as two more proteins with an apparent molecular mass of 26 and 19 kDa. Expression directed by this DNA fragment with various deletions suggested that the coding region for the two smaller proteins was contained in a 1-kb DNA region. Moreover, the size of both proteins was reduced by the same amount by an internal BglII-BglII DNA deletion, suggesting that they were translated from overlapping genes. Similar results were obtained with another independently cloned 6.1-kb Pseudomonas DNA, which in addition coded for a 31-kDa protein of opposite orientation. The nucleotide sequence of the 1-kb region above revealed an open reading frame with a signal sequence typical of secretory proteins and a potential in-phase internal translation initiation site. Pulse-chase and localization studies in E. coli showed that the 26-kDa protein was a precursor of a secreted periplasmic 23-kDa protein (PlcR1) while the 19-kDa protein (PlcR2) was mostly cytoplasmic. These results indicate the expression of Pseudomonas in-phase overlapping genes in E. coli.     

Identification of two new members of the CSMD gene family

CSMD1 is a putative suppressor of squamous cell carcinomas mapping to human chromosomal region 8p23. We have cloned two new members of this gene family, CSMD2 and CSMD3. The three CSMD proteins have very similar structures, each consisting of 14 CUB domains separated from one another by a sushi domain, an additional uninterrupted array of sushi domains, a single transmembrane domain, and a short cytoplasmic tail. CUB and sushi domains are thought to be sites of protein-protein or protein-ligand interactions, suggesting that CSMD proteins are either transmembrane receptors or adhesion proteins. The cytoplasmic tail sequences are highly conserved within the vertebrate lineage. CSMD2 maps to a chromosomal region that may contain a suppressor of oligodendrogliomas, yet its expression is elevated in some head and neck cancer cell lines. Functional overlap between the CSMD1 and the CSMD2 proteins may modify the phenotype resulting from the loss of either protein in tumors.     

Structure prediction of a complex between the chromosomal protein HMG-D and DNA

Non-histone chromosomal proteins are an important part of nuclear structure and function due to their ability to interact with DNA to form and modulate chromatin structure and regulate gene expression. However, the understanding of the function of chromosomal proteins at the molecular level has been hampered by the lack of structures of chromosomal protein–DNA complexes. We have carried out a molecular dynamics modeling study to provide insight into the mode of DNA binding to the chromosomal HMG-domain protein, HMG-D. Three models of a complex of HMG-D bound to DNA were derived through docking the protein to two different DNA fragments of known structure. Molecular dynamics simulations of the complexes provided data indicating the most favorable model. This model was further refined by molecular dynamics simulation and extensively analyzed. The structure of the corresponding HMG-D-DNA complex exhibits many features seen in the NMR structures of the sequence-specific HMG-domain-DNA complexes, lymphoid enhancer factor 1 (LEF-1) and testis determining factor (SRY). The model reveals differences from these known structures that suggest how chromosomal proteins bind to many different DNA sequences with comparable affinity. Proteins 30:113–135, 1998. © 1998 Wiley-Liss, Inc.     

Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli

Inhibitory effects of six antibiotics (kasugamycin, tetracycline, chloramphenicol, sparsomycin, puromycin and rifampicin) on the biosynthesis of envelope proteins of Escherichia coli were examined and compared with those on the biosynthesis of cytoplasmic proteins. Kasugamycin, puromycin and rifampicin were much more inhibitory to the over-all biosynthesis of cytoplasmic proteins than to that of envelope proteins. On the contrary, tetracycline and sparsomycin showed much stronger inhibitory effects on the biosynthesis of envelope proteins than on that of cytoplasmic proteins. Chloramphenicol showed little difference in its inhibitory effect on the biosynthesis of envelope proteins and cytoplasmic proteins.The envelope proteins were labeled with [³H]arginine in the presence of the antibiotics and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The inhibitory effects of the antibiotics on the biosynthesis of individual envelope proteins were then examined. Inhibition patterns were found to be widely different from one envelope protein to the other. For example, the biosynthesis of one major envelope protein of molecular weight 38,000 was more resistant to kasugamycin, chloramphenicol and sparsomycin than that of the other envelope proteins. On the other hand, the biosynthesis of another major envelope protein (lipoprotein) of about 7500 molecular weight was much more resistant to puromycin and rifampicin than that of the other envelope proteins. In the case of tetracycline, little differential inhibitory effect on the biosynthesis of individual envelope proteins was observed.Stability of messenger RNAs for individual envelope proteins was also determined from the inhibitory effect of rifampicin on their biosynthesis. It was found that the average of half lives of mRNAs for major envelope proteins examined (5.5 minutes) is twice as long as the average of those of mRNAs for cytoplasmic proteins (2 minutes), except for the lipoprotein of about 7500 molecular weight which has extremely stable mRNA with a half life of 11.5 minutes. From these results the envelope proteins of E. coli appear to be biosynthesized in a somewhat different manner from that of the cytoplasmic proteins. Furthermore, at least some envelope proteins may have their own specific biosynthetic systems.     

AUT1, a gene essential for autophagocytosis in the yeast Saccharomyces cerevisiae.

Autophagocytosis is a starvation-induced process responsible for transport of cytoplasmic proteins to the vacuole. In Saccharomyces cerevisiae, autophagy is characterized by the phenotypic appearance of autophagic vesicles inside the vacuole of strains deficient in proteinase yscB. The AUT1 gene, essential for autophagy, was isolated by complementation of the sporulation deficiency of a diploid aut1-1 mutant strain by a yeast genomic library and characterized. AUT1 is located on the right arm of chromosome XIV, 10 kb from the centromere, and encodes a protein of 310 amino acids, with an estimated molecular weight of 36 kDa. Cells carrying a chromosomal deletion of AUT1 are defective in the starvation-induced bulk flow transport of cytoplasmic proteins to the vacuole. aut1 null mutant strains are completely viable but show decreased survival rates during starvation. Homozygous delta aut1 diploid cells fail to sporulate. The selective cytoplasm-to-vacuole transport of aminopeptidase I is blocked in logarithmically growing and in starved delta autl cells. Deletion of the AUT1 gene had no obvious influence on secretion, fluid phase endocytosis, or vacuolar protein sorting. This supports the idea of autophagocytosis as being a novel route transporting proteins from the cytoplasm to the vacuole.     

Cellular compartmentalization of protein kinase activity during the cell cycle

The quantities and types of protein kinases found in the cytoplasmic and nuclear or chromosomal compartments of interphase and mitotic human culture cells were compared. Using histone as substrate, the total quantity of kinases recovered from cytoplasmic and chromosomal fractions of mitotic cells was several times greater than from cytoplasmic and nuclear fractions of interphase cells. In both mitotic and interphase cells, more activity was recovered from cytoplasmic fractions than from chromosomal or nuclear fractions, respectively. When activity against various substrates was examined, mitotic chromosomal extracts were found to display the greatest preference for the H1 fraction of histones. Neither cytoplasmic nor chromosomal fractions from mitotic cells exhibited enhanced activity in the presence of cAMP, whereas the activity of both cytoplasmic and nuclear fractions of interphase cells was enhanced. Protein kinases, previously identified by nondenaturing polyacrylamide gel electrophoresis as present in the cytoplasmic fraction of mitotic but not interphase cells, were also present in chromosomal fractions of mitotic cells; only one of these kinases may be present in nuclear extracts of interphase cells. In addition, the profiles of nuclear extracts of interphase cells differ from their cytoplasmic fractions. These results indicate that there are protein kinases which are restricted to the mitotic phase of the cell cycle and that they apparently partition between the cytoplasmic and chromosomal compartments of cells in mitosis.     

Cloning, mapping, and characterization of the Escherichia coli prc gene, which is involved in C-terminal processing of penicillin-binding protein 3.

The prc gene, which is involved in cleavage of the C-terminal peptide from the precursor form of penicillin-binding protein 3 (PBP 3) of Escherichia coli, was cloned and mapped at 40.4 min on the chromosome. The gene product was identified as a protein of about 80 kDa in maxicell and in vitro systems. Fractionation of the maxicells producing the product suggested that the product was associated with the periplasmic side of the cytoplasmic membrane. This was consistent with the notion that the C-terminal processing of PBP 3 probably occurs outside the cytoplasmic membrane: the processing was found to be dependent on the secY and secA functions, indicating that the prc product or PBP 3 or both share the translocation machinery with other extracytoplasmic proteins. DNA sequencing analysis of the prc gene region identified an open reading frame, with two possible translational starts 6 bp apart from each other, that could code for a product with a calculated molecular weight of 76,667 or 76,432. The prc mutant was sensitive to thermal and osmotic stresses. Southern analysis of the chromosomal DNA of the mutant unexpectedly revealed that the mutation was a deletion of the entire prc gene and thus that the prc gene is conditionally dispensable. The mutation resulted in greatly reduced heat shock response at low osmolarity and in leakage of periplasmic proteins.     

The occurrence of extended acidic sequences in nonhistone chromosomal proteins

Nonhistone chromosomal proteins and soluble cytoplasmic proteins from rat liver were treated with a combination of proteases and chemical reagents which split a variety of peptide bonds but do not attack sequences consisting predominantly or exclusively of acidic amino acid residues. Analysis of the resulting digests by gel filtration chromatography and column electrophoresis demonstrated that, relative to cytoplasmic proteins, nonhistone chromosomal proteins are rich in highly charged, acidic peptides up to 12 residues in length, but rarely contain very long peptides consisting exclusively of acidic residues such as are found in the nonhistone chromosomal proteins HMG1 and HMG2.     

NUCLEIC ACID AND PROTEIN METABOLISM DURING THE MITOTIC CYCLE IN VICIA FABA

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C¹⁴ and uridine-H³ were used as ribonucleic acid (RNA) precursors, thymidine-H³ as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C¹⁴ as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA.     

Identification of novel nuclear localization signals of Drosophila myeloid leukemia factor

Myeloid leukemia factor 1 (MLF1) was first identified as part of a leukemic fusion protein produced by a chromosomal translocation, and MLF family proteins are present in many animals. In mammalian cells, MLF1 has been described as mainly cytoplasmic, but in Drosophila, one of the dMLF isoforms (dMLFA) localized mainly in the nucleus while the other isoform (dMLFB), that appears to be produced by the alternative splicing, displays both nuclear and cytoplasmic localization. To investigate the difference in subcellular localization between MLF family members, we examined the subcellular localization of deletion mutants of dMLFA isoform. The analyses showed that the C-terminal 40 amino acid region of dMLFA is necessary and sufficient for nuclear localization. Based on amino acid sequences, we hypothesized that two nuclear localization signals (NLSs) are present within the region. Site-directed mutagenesis of critical residues within the two putative NLSs leads to loss of nuclear localization, suggesting that both NLS motifs are necessary for nuclear localization.     

Dimerization of the scaffolding protein ZO-1 through the second PDZ domain

The tight junction protein ZO-1 is known to link the transmembrane proteins occludin, claudins, and JAMs to many cytoplasmic proteins and the actin cytoskeleton. Although specific roles for ZO-1 at the tight junction are unknown, it is widely assumed that ZO-1, together with its homologs ZO-2 and ZO-3, serves as a platform to scaffold various transmembrane and cytoplasmic tight junction proteins. Thus the manner in which the zonula occludens (ZO) proteins multimerize has implications for the protein networks they can coordinate. The purpose of our study was to determine whether ZO-1 forms homodimers and to determine the protein interaction region. Using laser light scattering and analytical centrifugation, we show that protein sequences corresponding to the NH(2)-terminal half of ZO-1 form stable homodimers with a submicromolar equilibrium dissociation constant. Analysis of the molecular weight of different truncated forms of ZO-1 revealed that the second PDZ domain is both necessary and sufficient for dimerization. This interaction does not use the beta-finger motif described for other PDZ dimers. Furthermore, ZO-1 does not dimerize via an Src homology 3 to Guk domain interaction as was demonstrated previously for MAGUKs, like PSD-95. Results from immunoprecipitation experiments with polarized Madin-Darby canine kidney epithelial cells stably transfected with full-length GFP-ZO-1 indicate that a substantial portion of ZO-1 forms homodimers in vivo. As described previously, ZO-1 also forms heterodimers with ZO-2 and ZO-3. We conclude that the dimerization of ZO proteins is unlike that of other MAGUKs and that the previously unrecognized ZO-1 homodimers may allow formation of protein networks distinct from those of heterodimers with ZO-2 and ZO-3.     

Inducible expression of the P, V, and NP genes of the paramyxovirus simian virus 5 in cell lines and an examination of NP-P and NP-V interactions.

The P, V, and NP genes of the paramyxovirus simian virus 5 (SV5) were cloned such that their expression was regulated by the tetracycline-controlled transactivator (M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992), and mammalian cell lines that inducibly expressed individually the P, V, or NP protein or coexpressed the P plus NP or V plus NP proteins were isolated. A plasmid that expresses the tetracycline-controlled transactivator linked, via the foot-and-mouth disease virus 2A cleavage peptide sequence, to the neomycin aminoglycoside phosphotransferase gene was constructed. Cells were cotransfected with this plasmid, and the appropriate responder plasmids and clonies were selected on the basis of their resistance to Geneticin (via the neomycin aminoglycoside phosphotransferase gene). The properties of these cell lines, in terms of the induction of the P, V, and NP genes, are described in detail. Both the P and V proteins were phosphorylated when expressed alone. In immunoprecipitation studies using a monoclonal antibody that recognizes both the P and V proteins, a nonphosphorylated host cell protein with an estimated molecular weight of 150,000 was coprecipitated with V but not P. Immunofluorescence data demonstrated that when expressed separately, the P protein had a diffuse cytoplasmic distribution, but the related V protein had both a nuclear and cytoplasmic distribution. The NP protein had a granular cytoplasmic distribution, giving rise to punctate and granular fluorescence. Coexpression of the NP and P proteins resulted in the accumulation of large cytoplasmic inclusion aggregates, similar to those visualized at late times in SV5-infected cells. Coexpression of V with NP led to a partial redistribution of the NP protein in that the NP protein had both a diffuse cytoplasmic and nuclear distribution in the presence of V, but no NP-V aggregates or inclusion bodies were visualized. Direct binding studies also revealed that NP bound to both P and V. For SV5, these studies suggest that V may have a role in keeping NP soluble prior to encapsidation.