The retinoblastoma protein {pll0 nB) interacts with many cellular proteins in complexes potentially important for its growth-suppressing [unction. We have developed and used an improved version of the yeast two-hybrid system to isolate human cDNAs encoding proteins able to bind pll0 RB. One clone encodes a novel type 1 protein phosphatase catalytic subunit (PP-la2), which differs from the originally defined PP-lc~ by an amino-terminal l 1-amino-acid insert. In vitro-binding assays demonstrated that PP-lc~ isoforms preferentially bind the hypophosphorylated form of p ll0 RB. Moreover, similar pll0 RB sequences are required for binding PP-lc~2 and SV40 large T antigen. Cell cycle synchrony experiments revealed that this association occurs from mitosis to early Gv The implications of these findings on the regulation of both proteins are discussed.

The bacterial stringent response serves as a paradigm for understanding global regulatory processes. It can be triggered by nutrient downshifts or starvation and is characterized by a rapid RelA-dependent increase in the alarmone (p)ppGpp. One hallmark of the response is the switch from maximum-growth-promoting to biosynthesis-related gene expression. However, the global transcription patterns accompanying the stringent response in Escherichia coli have not been analyzed comprehensively. Here, we present a time series of gene expression profiles for two serine hydroxymate-treated cultures: (i) MG1655, a wild-type E. coli K-12 strain, and (ii) an isogenic relA⌬251 derivative defective in the stringent response. The stringent response in MG1655 develops in a hierarchical manner, ultimately involving almost 500 differentially expressed genes, while the relA⌬251 mutant response is both delayed and limited in scope. We show that in addition to the downregulation of stable RNA-encoding genes, flagellar and chemotaxis gene expression is also under stringent control. Reduced transcription of these systems, as well as metabolic and transporter-encoding genes, constitutes much of the down-regulated expression pattern. Conversely, a significantly larger number of genes are up-regulated. Under the conditions used, induction of amino acid biosynthetic genes is limited to the leader sequences of attenuator-regulated operons. Instead, up-regulated genes with known functions, including both regulators (e.g., rpoE, rpoH, and rpoS) and effectors, are largely involved in stress responses. However, one-half of the up-regulated genes have unknown functions. How these results are correlated with the various effects of (p)ppGpp (in particular, RNA polymerase redistribution) is discussed.

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, Apl2, is expressed predominantly at the G1-S boundary and in the S phase of the cell cycle. The nucleotide sequence of Apl2 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 ofApl2 in mammalian cells significantly enhances E2F-dependent transcriptional activity. These results suggest that Apl2 encodes a protein with properties known to be characteristic of transcription factor E2F.The retinoblastoma gene (RB), the first tumor suppressor gene identified, encodes a nuclear phosphoprotein which is ubiquitously expressed in vertebrates (19,20,42,44 (45,47), fluctuates with the cell cycle (7, 11, 13); (ii) the unphosphorylated form of RB is present predominantly in the Go-G1 stage (11, 13); (iii) microinjection of unphosphorylated RB into cells at early G1 inhibits their progression into the S phase (22). These data suggest that RB serves as a critical regulator of entry into the cell cycle and that its inactivation in normal cells leads to deregulated growth.How RB functions is the subject of intense inquiry. Two known biochemical properties of the RB protein have been described; one is its intrinsic DNA-binding activity, which was mapped to its C-terminal 300 amino acid residues (44, 66); another is its ability to interact with several oncoproteins of DNA tumor viruses (12,16,67). This interaction was mapped to two discontinuous regions at amino acids 394 to 571 and 649 to 773, designated as the T-binding domains (29,33). Interestingly, mutations of RB protein in tumors were *

Escherichia coli DH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStar's SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion of leuLABCD and harbors both the relA1 and spoT1 alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence of deoR is wild type, necessitating reexamination of the assumed basis for the high transformability of DH10B.Molecular biology studies rely heavily on Escherichia coli for essential operations, ranging from the simple propagation of plasmid DNA to the creation of large clone libraries for wholegenome sequence determination. Among the strains developed as hosts for these everyday applications, DH10B (17) is commonly used across the research community, taking advantage of particularly useful properties exhibited by the strain. These include high transformation efficiency, the ability to take up and stably maintain large plasmids, the lack of methylation-dependent restriction systems (MDRS), and colony screening via lacZ-based ␣-complementation. However, analysis of sequenced bacterial artificial chromosome (BAC) clones derived from DH10B shows a high incidence of insertion sequence (IS) transposition from the chromosome into the cloned fragment (25).The genome of DH10B was constructed before the modern era of molecular biology, through a series of genetic manipulations (Fig. 1). The progenitors were all K-12 strains, with the exception of D7091F, in which a region surrounding the ⌬(araA-leu)7697 deletion had been derived from E. coli B SB3118 by P1 transduction (John Wertz, personal communication). Ultimately, MC1061 (9) served as a starting point for Hanahan and coworkers to replace alleles by using a series of P1 transductions that resulted in DH10B (17). Among the engineered gene replacements were recA1 ...

By exploring global gene expression of Escherichia coli growing on six different carbon sources, we discovered a striking genome transcription pattern: as carbon substrate quality declines, cells systematically increase the number of genes expressed. Gene induction occurs in a hierarchical manner and includes many factors for uptake and metabolism of better but currently unavailable carbon sources. Concomitantly, cells also increase their motility. Thus, as the growth potential of the environment decreases, cells appear to devote progressively more energy on the mere possibility of improving conditions. This adaptation is not what would be predicated by classic regulatory models alone. We also observe an inverse correlation between gene activation and rRNA synthesis suggesting that reapportioning RNA polymerase (RNAP) contributes to the expanded genome activation. Significant differences in RNAP distribution in vivo, monitored using an RNAP-green fluorescent protein fusion, from energy-rich and energy-poor carbon source cultures support this hypothesis. Together, these findings represent the integration of both substrate-specific and global regulatory systems, and may be a bacterial approximation to metazoan risk-prone foraging behavior.Jacob and Monod originally studied Escherichia coli gene regulation using carbon catabolism as the experimental system (specifically lactose). They elucidated that genes needed to metabolize lactose are specifically induced by that substrate (1). Over the ensuing 40 years this model has been refined and extended to many substrates and it is now generally accepted that virtually all carbohydrate catabolic genes can be regulated by substrate-specific induction (2). This mechanism is attractive because it promotes efficient use of cellular resources, and energy need not be wasted producing enzymes and transporters for substrates unless they are available.In addition to these highly specific mechanisms, cells also have several levels of global regulation. A prime example is carbon catabolite repression, a multifactorial system that blocks expression of alternative carbon utilization pathways when glucose is present (3). Relief from carbon catabolite repression, among other effects, activates the cyclic AMP receptor protein (CRP), 1 a global transcription factor that positively regulates most carbon catabolic pathways including those for glucose (4). Such global mechanisms have the advantage of responding to many different conditions and can potentiate a spectrum of transcriptional outcomes.While much is known about how individual substrate-specific and global systems regulate a limited set of operons under a defined condition, much less is known regarding how the two coordinate genome-wide transcription under a range of conditions. In this paper, we report the findings from a global gene expression study of E. coli growing on a series of six carbon sources. A transcription pattern emerges from these profiles that expands hierarchically as the growth rate declines. Inspection of the u...