Structural and functional studies of the early T lymphocyte activation 1 (Eta-1) gene. Definition of a novel T cell-dependent response associated with genetic resistance to bacterial infection.

We describe a murine cDNA, designated Early T lymphocyte activation 1 (ETA-1) which is abundantly expressed after activation of T cells. Eta-1 encodes a highly acidic secreted product having structural features of proteins that bind to cellular adhesion receptors. The Eta-1 gene maps to a locus on murine chromosome 5 termed Ric that confers resistance to infection by Rickettsia tsutsugamushi (RT), an obligate intracellular bacterium that is the etiological agent for human scrub typhus. With one exception, inbred mouse strains that expressed the Eta-1a allele were resistant to RT infection (RicR), and inbred strains expressing the Eta-1b allele were susceptible (RicS). These findings suggest that Eta-1 is the gene inferred from previous studies of the Ric locus (5). Genetic resistance to RT infection is associated with a strong Eta-1 response in vivo and inhibition of early bacterial replication. Eta-1 gene expression appears to be part of a surprisingly rapid T cell-dependent response to bacterial infection that may precede classical forms of T cell-dependent immunity.

The amino-terminal region of the retinoblastoma gene product binds a novel nuclear matrix protein that co-localizes to centers for RNA processing.

The tumor suppressing capacity of the retinoblastoma protein (p110RB) is dependent on interactions made with cellular proteins through its carboxy-terminal domains. How the p110RB amino-terminal region contributes to this activity is unclear, though evidence now indicates it is important for both growth suppression and regulation of the full-length protein. We have used the yeast two-hybrid system to screen for cellular proteins which bind to the first 300 amino acids of p110RB. The only gene isolated from this screen encodes a novel 84-kD nuclear matrix protein that localizes to subnuclear regions associated with RNA processing. This protein, p84, requires a structurally defined domain in the amino terminus of p110RB for binding. Furthermore, both in vivo and in vitro experiments demonstrate that p84 binds preferentially to the functionally active, hypophosphorylated form of p110RB. Thus, the amino terminus of p110RB may function in part to facilitate the binding of growth promoting factors at subnuclear regions actively involved in RNA metabolism.

Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F.

The retinoblastoma protein interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function. To identify these proteins, we isolated nine distinct clones by direct screening of cDNA expression libraries using purified RB protein as a probe. One of these clones, Ap12, is expressed predominantly at the G1-S boundary and in the S phase of the cell cycle. The nucleotide sequence of Ap12 has features characteristic of transcription factors. The C-terminal region binds to unphosphorylated RB in regions similar to those to which T antigen binds and contains a transactivation domain. A region containing a potential leucine zipper flanked by basic residues is able to bind an E2F recognition sequence specifically. Expression of Ap12 in mammalian cells significantly enhances E2F-dependent transcriptional activity. These results suggest that Ap12 encodes a protein with properties known to be characteristic of transcription factor E2F.

RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein.

Unlike mammalian and yeast cells, little is known about how plants regulate G1 progression and entry into the S phase of the cell cycle. In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB). In contrast, G1 control in Saccharomyces cerevisiae does not utilize an RB-like protein. We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins. Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini. At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex. RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin. These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function. RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB. RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication. These results suggest that G1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.

Mutational analysis of STE5 in the yeast Saccharomyces cerevisiae: application of a differential interaction trap assay for examining protein-protein interactions.

Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold that organizes the components of the mitogen-activated protein kinase (MAPK) cascade. A new method was developed to isolate missense mutations in Ste5 that differentially affect the ability of Ste5 to interact with either of two MAPK cascade constituents, the MEKK (Ste11) and the MEK (Ste7). Mutations that affect association with Ste7 or with Ste11 delineate discrete regions of Ste5 that are critical for each interaction. Co-immunoprecipitation analysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G protein beta subunit), and Fus3 (MAPK), confirmed that each mutation specifically affects the interaction of Ste5 with only one protein. When expressed in a ste5 delta cell, mutant Ste5 proteins that are defective in their ability to interact with either Ste11 or Ste7 result in a markedly reduced mating proficiency. One mutation that clearly weakened (but did not eliminate) interaction of Ste5 with Ste7 permitted mating at wild-type efficiency, indicating that an efficacious signal is generated even when Ste5 associates with only a small fraction of (or only transiently with) Ste7. Ste5 mutants defective in association with Ste11 or Ste7 showed strong interallelic complementation when co-expressed, suggesting that the functional form of Ste5 in vivo is an oligomer.

Definition of T-cell specific DNA-binding factors that interact with a 3'-silencer in the CD4+ T-cell gene Rpt-1.

Analysis of the region 3' to the CD4+ T-cell gene Rpt-1 (encoding regulatory protein T-lymphocyte 1) led to the definition of a silencer element that inhibits heterologous gene expression in certain CD4+ T-cell lines but not in B-cell or non-lymphoid cell lines. Functional silencer activity in vivo was associated with the presence of a specific silencer-DNA-protein complex in electrophoretic mobility shift assays with T-cell extracts. Formation of this complex was selectively inhibited by the region in HIV-1 containing a silencer element. We discuss the possibility that DNA-binding factors may coregulate HIV-1 and Rpt-1 gene expression through a common transcriptional silencer element.

Disruption of RB/E2F-1 interaction by single point mutations in E2F-1 enhances S-phase entry and apoptosis.

The retinoblastoma protein (RB) has been proposed to function as a negative regulator of cell proliferation by complexing with cellular proteins such as the transcription factor E2F. To study the biological consequences of the RB/E2F-1 interaction, point mutants of E2F-1 which fail to bind to RB were isolated by using the yeast two-hybrid system. Sequence analysis revealed that within the minimal 18-amino acid peptide of E2F-1 required for RB binding, five residues, Tyr (position 411), Glu (419), and Asp-Leu-Phe (423-425), are critical. These amino acids are conserved among the known E2F family members. While mutation of any of these five amino acids abolished binding to RB, all mutants retained their full transactivation potential. Expression of mutated E2F-1, when compared with that of wild-type, significantly accelerated entry into S phase and subsequent apoptosis. These results provide direct genetic evidence for the biological significance of the RB/E2F interaction and strongly suggest that the interplay between RB and E2F is critical for proper cell cycle progression.

Retinoblastoma-related proteins in plants: homologues or orthologues of their metazoan counterparts?

The mammalian retinoblastoma tumor suppressor protein (pRb) regulates cell division, differentiation and apoptotic pathways in specific cell types. In association with other proteins, pRb acts in part by modulating transcriptional activity. Elements of the pRb regulatory network have been identified in higher plants. Recent findings involving these proteins, which display amino acid sequence homology and biochemical binding properties analogous to their mammalian counterparts, are discussed.

Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157:H7: Shiga toxin as a phage late-gene product.

Lysogenic bacteriophages are major vehicles for the transfer of genetic information between bacteria, including pathogenicity and/or virulence determinants. In the enteric pathogen Escherichia coli O157:H7, which causes hemorrhagic colitis and hemolytic-uremic syndrome, Shiga toxins 1 and 2 (Stx1 and Stx2) are phage encoded. The sequence and analysis of the Stx2 phage 933W is presented here. We find evidence that the toxin genes are part of a late-phage transcript, suggesting that toxin production may be coupled with, if not dependent upon, phage release during lytic growth. Another phage gene, stk, encodes a product resembling eukaryotic serine/threonine protein kinases. Based on its position in the sequence, Stk may be produced by the prophage in the lysogenic state, and, like the YpkA protein of Yersinia species, it may interfere with the signal transduction pathway of the mammalian host. Three novel tRNA genes present in the phage genome may serve to increase the availability of rare tRNA species associated with efficient expression of pathogenicity determinants: both the Shiga toxin and serine/threonine kinase genes contain rare isoleucine and arginine codons. 933W also has homology to lom, encoding a member of a family of outer membrane proteins associated with virulence by conferring the ability to survive in macrophages, and bor, implicated in serum resistance.

New tools for protein linkage mapping and general two-hybrid screening.

The two-hybrid system has proved to be a facile method for detecting and analyzing protein-protein interactions. An expanded application of this system, protein linkage mapping, provides a means of identifying interactions on a global scale and should prove a powerful tool in analyzing whole genomes as their sequences become available. To overcome some of the inherent difficulties in such a large-scale approach, we have constructed a set of new strains and vectors that will allow for more efficient screening. The strains contain a GAL1-URA3 reporter for positive and negative selection, as well as a UAS(G)-lacZ reporter. The strains are of opposite mating types, permitting libraries present in one strain to be easily screened against a second library in the companion strain. We also constructed a family of CEN-based vectors for expression of both Gal4 DNA-binding and activation domain fusions. These plasmids include a hemagglutinin epitope tag and different polylinkers to increase the ease of subcloning. CEN-based vectors are maintained at 1-2 copies per cell, limiting the number of individual cells containing multiple plasmids that can confuse further analyses, and ensuring that fusions are not expressed at toxic levels. Using these vectors, both homo- and heterodimeric interactions resulted in up to 10-fold higher reporter gene transcription than obtained with 2micro;-based plasmids, despite significantly lower protein levels. In addition to protein linkage mapping, these reagents should be generally useful in standard two-hybrid applications.

TOUSLED is a nuclear serine/threonine protein kinase that requires a coiled-coil region for oligomerization and catalytic activity.

The TOUSLED (TSL) gene is essential for the proper morphogenesis of leaves and flowers in Arabidopsis thaliana. Protein sequence analysis predicts TSL is composed of a carboxyl-terminal protein kinase catalytic domain and a large amino-terminal regulatory domain. TSL fusion proteins, expressed in and purified from yeast, were used to demonstrate TSL protein kinase activity in vitro. TSL trans-autophosphorylates on serine and threonine residues, and phosphorylates exogenous substrates. Using the yeast two-hybrid system, TSL was found to oligomerize via its NH2-terminal domain. A deletion series indicates that a region containing two alpha-helical segments predicted to participate in a coiled-coil structure is essential for oligomerization. TSL localizes to the nucleus in plant cells through an essential NH2-terminal nuclear localization signal; however, this signal is not necessary for protein kinase activity. Finally, deletion mutants demonstrate a strict correlation between catalytic activity and the ability to oligomerize, arguing that activation of the protein kinase requires interaction between TSL molecules.

Identification of a human homologue of yeast nuc2 which interacts with the retinoblastoma protein in a specific manner.

The full-length cDNA clone which encodes a novel 824-amino acid protein was characterized. The predicted protein contains ten 34-amino acid repeats characteristic of the tetratricopeptide repeat protein family. The sequence homology and organization of the 10 repeats are similar to those of the nuc2 protein of fission yeast and bimA protein of Aspergillus, which suggests that the newly identified protein could be the human homologue of nuc2 (H-NUC). Consistent with this notion, the M(r) 95,000 H-NUC is a nuclear protein with DNA binding activity. This protein binds to hypophosphorylated Rb protein in a region indistinguishable from that to which SV40 large T antigen binds. However, Rb also binds to H-NUC at the tetratricopeptide repeat motif, a region which contains sequences different from the binding motifs of either T-antigen or E2F-1. To mimic the temperature-sensitive mutant of yeast nuc2, an H-NUC mutant was made in which the highly conserved glycine 640 residue was changed to aspartic acid. In contrast to wild-type H-NUC, the mutant was temperature sensitive in binding to Rb protein. These results, taken together, suggest that the interaction between H-NUC and Rb may be significant.

Identification of discrete structural domains in the retinoblastoma protein. Amino-terminal domain is required for its oligomerization.

To characterize the protein product of the retinoblastoma tumor suppressor gene biochemically, a recombinant human protein was produced in an Escherichia coli expression system. The full-length protein, p110RB, and an amino-terminal truncated form, p56RB, were expressed and purified to near homogeneity by conventional chromatographic procedures. To probe the structural organization of the retinoblastoma protein the purified proteins were subjected to partial proteolysis by trypsin, chymotrypsin, and subtilisin. Four discrete structural domains were revealed in p110RB by this method. Two of these structural domains, found in both p56RB and p110RB, were mapped to the carboxyl-terminal half of the protein and corresponded to the SV40 large T binding domains defined previously by genetic methods. In addition two distinct domains in the amino-terminal half of the protein were also defined. A potential role for these newly defined amino-terminal domains was uncovered upon analysis of the purified proteins by nondenaturing polyacrylamide gel electrophoresis. p110RB revealed multiple bands by this method, suggesting the formation of oligomeric structures by the protein, while this property was not observed for p56RB. Electron microscopy of p110RB revealed linearly extended, macromolecular structures, further supporting the formation of homologous higher order structures by the full-length retinoblastoma protein. Analysis of the interactions between retinoblastoma protein molecules using the yeast two-hybrid system confirmed that the retinoblastoma protein could self-associate and that this association was mediated by interactions between the amino- and carboxyl-terminal ends of the protein. These observations suggest that the retinoblastoma protein contains multiple structural domains with the amino-terminal domains being required for oligomerization of the full-length protein.

The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit.

The retinoblastoma protein (p110RB) interacts with many cellular proteins in complexes potentially important for its growth-suppressing function. We have developed and used an improved version of the yeast two-hybrid system to isolate human cDNAs encoding proteins able to bind p110RB. One clone encodes a novel type 1 protein phosphatase catalytic subunit (PP-1 alpha 2), which differs from the originally defined PP-1 alpha by an amino-terminal 11-amino-acid insert. In vitro-binding assays demonstrated that PP-1 alpha isoforms preferentially bind the hypophosphorylated form of p110RB. Moreover, similar p110RB sequences are required for binding PP-1 alpha 2 and SV40 large T antigen. Cell cycle synchrony experiments revealed that this association occurs from mitosis to early G1. The implications of these findings on the regulation of both proteins are discussed.

Analysis and comparison of the mouse and human immunoglobulin heavy chain JH-Cmu-Cdelta locus.

We report here 23,686 bases of contiguous DNA sequences from the mouse germline immunoglobulin heavy chain (H) constant (C) mu delta region. The sequence spans the joining (JH) regions, the mu constant region (C mu), the delta constant region (C delta) coding regions, a domain relic, the mu switch region (S mu), seven blocks of simple sequence repeats, a large unique sequence inverted repeat, a large unique sequence forward repeat, and all of the intervening material. A comparison of this 23.7-kb region with the corresponding human C mu/C delta region reveals clear homology in the coding and introns of C mu but not in the 5' flanking J gene segments nor in the intergenic and C delta regions. This mixed pattern of similarity between the human and the mouse sequences contrasts with high levels of similarity found in the T-cell receptor C alpha/C delta region and alpha and beta myosin genes and the very low levels found in the gamma-crystallin, XRCC1, and beta-globin gene clusters. The human and mouse comparison further suggests the incorporation of novel sequences into expressed genes of IgD.

A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants.

Geminiviruses replicate in nuclei of mature plant cells after inducing the accumulation of host DNA replication machinery. Earlier studies showed that the viral replication factor, AL1, is sufficient for host induction and interacts with the cell cycle regulator, retinoblastoma (pRb). Unlike other DNA virus proteins, AL1 does not contain the pRb binding consensus, LXCXE, and interacts with plant pRb homo logues (pRBR) through a novel amino acid sequence. We mapped the pRBR binding domain of AL1 between amino acids 101 and 180 and identified two mutants that are differentially impacted for AL1-pRBR interactions. Plants infected with the E-N140 mutant, which is wild-type for pRBR binding, developed wild-type symptoms and accumulated viral DNA and AL1 protein in epidermal, mesophyll and vascular cells of mature leaves. Plants inoculated with the KEE146 mutant, which retains 16% pRBR binding activity, only developed chlorosis along the veins, and viral DNA, AL1 protein and the host DNA synthesis factor, proliferating cell nuclear antigen, were localized to vascular tissue. These results established the importance of AL1-pRBR interactions during geminivirus infection of plants.