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In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fMet-tRNA fMet . Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entrance channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Patient-reported outcomes (PRO) are increasingly utilized as part of the routine clinical assessment in people with multiple sclerosis (pwMS). The long-term effect of disease modifying therapies (DMTs) and their discontinuation on PRO measures remains largely unknown., Methods: Two pwMS groups treated with natalizumab were selected from the New York State MS Consortium (NYSMSC) database. The first group utilized long-term follow-up data of pwMS that either still continue natalizumab treatment or discontinued. Minimal requirement of three visits (before natalizumab initiation, during treatment and after discontinuation/latest follow-up) was implemented. The second group consisted of pwMS that completed PRO questionnaire on the day of the infusion and 7 days later PROs were assessed using the LIFEware System™ that assesses limitations in multiple physical and psychosocial domains. Additional physical disability was assessed using Expanded Disability Status Scale (EDSS) and Timed 25-ft walk test (T25FWT). PRO reports were Rasch-transformed, ranging from 0 to 100, with higher scores indicating a better outcome. Linear mixed-effect models and paired analyses were utilized., Results: Within the prospective cohort, 242 pwMS were followed on average of 6.5 years. Greater number of PRO domains worsened in the 141 pwMS that discontinued natalizumab when compared to 101 pwMS that remained on the drug (10 vs. 2 PRO domains). PwMS that discontinued natalizumab had significant decline in PROs regarding lower extremities, bladder and bower control and psychosocial aspects (feeling lonesome). Contrarily, pwMS that continued natalizumab had significant improvement in bladder and bowel PRO measures. Seven days after the natalizumab infusion, the 67 pwMS from the prospective cohort reported improvement in PRO measures of fatigue (62.8 vs. 66.4, p = 0.019), bladder limitations (80.3 vs. 85.0, p = 0.012), and feelings of lonesomeness (81.2 vs. 88.0, p = 0.009)., Conclusion: Continuous natalizumab treatment provides long-term stability or improvement in PRO measures. Natalizumab also provides short term improvements recorded after the infusion., (Copyright © 2023 Elsevier B.V. All rights reserved.)

NusG is a conserved regulatory protein that interacts with elongation complexes (ECs) of RNA polymerase, DNA, and RNA to modulate transcription in multiple and sometimes opposite ways. In Escherichia coli, NusG suppresses pausing and increases elongation rate, enhances termination by E. coli rho and phage HK022 Nun protein, and promotes antitermination by lambdaN and in ribosomal RNA operons. We report NMR studies that suggest that E. coli NusG consists of two largely independent N- and C-terminal structural domains, NTD and CTD, respectively. Based on tests of the functions of the NTD and CTD and variants of NusG in vivo and in vitro, we find that NTD alone is sufficient to suppress pausing and enhance transcript elongation in vitro. However, neither domain alone can enhance rho-dependent termination or support antitermination, indicating that interactions of both domains with ECs are required for these processes. We propose that the two domains of NusG mediate distinct interactions with ECs: the NTD interacts with RNA polymerase and the CTD interacts with rho and other regulators, providing NusG with different combinations of interactions to effect different regulatory outcomes.

Although retinol bound to retinol-binding protein (RBP) is the most abundant retinoid form present in the circulations of humans and most mammals, other retinoid and proretinoid forms are also present in the blood. We are interested in understanding to what extent each of these circulating retinoid forms contributes towards retinoid actions within cells and tissues. Here we report two studies focused on this question. First, we examined retinoid transport and storage in RBP-deficient mice that lack circulating RBP. These mice under normal laboratory conditions are phenotypically normal except for a visual impairment early in life that is corrected if the mice are maintained on a vitamin A-sufficient diet throughout life. The RBP-deficient mice take up vitamin A from the diet into most tissues at least as well as wild type mice. Compared to wild type mice, mice lacking RBP accumulate excess vitamin A in the liver, since there is no RBP to facilitate mobilization of stored retinol from hepatic stores. In a second study, we explored in vitro the actions of carotene cleavage enzyme (CCE) in facilitating beta-carotene cleavage to retinoid in the testis. CCE is most highly expressed in the testis. Pull-down experiments coupled with MALDI-MS analysis showed that mouse testis CCE is able to interact with the testis-specific lactate dehydrogenase-C (LDH-C) isoform. This may suggest that CCE and LDH-C act in concert to catalyze beta-carotene cleavage.

cAMP-dependent protein kinase is targeted to discrete subcellular locations by a family of specific anchor proteins (A-kinase anchor proteins, AKAPs). Localization recruits protein kinase A (PKA) holoenzyme close to its substrate/effector proteins, directing and amplifying the biological effects of cAMP signaling.AKAPs include two conserved structural modules: (i) a targeting domain that serves as a scaffold and membrane anchor; and (ii) a tethering domain that interacts with PKA regulatory subunits. Alternative splicing can shuffle targeting and tethering domains to generate a variety of AKAPs with different targeting specificity. Although AKAPs have been identified on the basis of their interaction with PKA, they also bind other signaling molecules, mainly phosphatases and kinases, that regulate AKAP targeting and activate other signal transduction pathways. We suggest that AKAP forms a "transduceosome" by acting as an autonomous multivalent scaffold that assembles and integrates signals derived from multiple pathways. The transduceosome amplifies cAMP and other signals locally and, by stabilizing and reducing the basal activity of PKA, it also exerts long-distance effects. The AKAP transduceosome thus optimizes the amplitude and the signal/noise ratio of cAMP-PKA stimuli travelling from the membrane to the nucleus and other subcellular compartments., (Copyright 2001 Academic Press.)

The study of bacteriophage lambda has provided key insights into fundamental biological processes. This review recalls some highlights in the history of lambda research, and relates how simple (but elegant) experiments yielded major scientific breakthroughs. What we know about recombination, gene regulation, and protein folding, for example, derives in large part from bacteriophage lambda genetics. Lambda not only represents a model system of scientific logic in a technology-driven age, but continues to reveal new principles of molecular biology., (Copyright 1999 Academic Press.)

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which confers resistance to many chemotherapeutic drugs. Monoclonal antibodies raised against P-gp have been used as tools to study P-gp topology and activity. Monoclonal antibody UIC2 recognizes a functional conformation of P-gp on the cell surface and blocks P-gp-mediated drug transport. Knowledge about the UIC2 epitope and the mechanism of its inhibitory effects may be helpful for understanding P-gp structure and developing P-gp inhibitors. In the present work, using several chimeras of MDR1 and MDR2, we found that the native sequence of the predicted extracellular loop between transmembrane domains (TM) 5 and 6 of P-gp is necessary, but not sufficient, for UIC2 reactivity. In addition, UIC2 reactivity is also affected by mutations in TM6, a region known to be involved in interactions of P-gp with substrates. These observations suggest that residues in the extracellular loop between TM5 and TM6 are directly involved in the display of the UIC2 epitope. Since TM6 has been shown to be actively involved in drug transport process, the proximity of this region to TM6 may help to explain why UIC2 binding is sensitive to the functional state of P-gp and why binding of UIC2 inhibits P-gp-mediated drug transport., (Copyright 1999 Academic Press.)

To determine whether the multidrug resistance gene MDR1 could act as a selectable marker in human subjects, we studied engraftment of peripheral blood progenitor cells (PBPCs) transduced with either MDR1 or the bacterial NeoR gene in six breast cancer patients. This study differed from previous MDR1 gene therapy studies in that patients received only PBPCs incubated in retroviral supernatants (no nonmanipulated PBPCs were infused), transduction of PBPCs was supported with autologous bone marrow stroma without additional cytokines, and a control gene (NeoR) was used for comparison with MDR1. Transduced PBPCs were infused after high-dose alkylating agent therapy and before chemotherapy with MDR-substrate drugs. We found that hematopoietic reconstitution can occur using only PBPCs incubated ex vivo, that the MDR1 gene product may play a role in engraftment, and that chemotherapy may selectively expand MDR1 gene-transduced hematopoietic cells relative to NeoR transduced cells in some patients.

Amyloid fibrils derived from the Japanese, Portuguese, and Swedish types of familial amyloidotic polyneuropathy all consist of a variant transthyretin (TTR) with a substitution of methionine for valine at position 30 (TTR Met 30). In an attempt to establish an animal model of TTR Met-30-associated homozygous familial amyloidotic polyneuropathy and to study the structural and functional properties of human TTR Met 30, we generated a mouse line carrying a null mutation at the endogenous ttr locus (ttr-/-) and the human mutant ttr gene (6.0-hMet 30) as a transgene. In these mice, human TTR Met-30-derived amyloid deposits were first observed in the esophagus and stomach when the mice were 11 months of age. With advancing age, amyloid deposits extended to various other tissues. Because no significant difference was detected in the onset, progression, and tissue distribution of amyloid deposition between the ttr-/- and ttr+/+ transgenic mice expressing 6.0-hMet 30, endogenous normal mouse TTR probably does not affect the deposition of human TTR Met-30-derived amyloid in mice. TTR is a tetramer composed of four identical subunits that binds thyroxine (T4) and plasma retinol-binding protein. The introduction of 6.0-hMet 30 into the ttr-/- mice significantly increased their depressed serum levels of T4 and retinol-binding protein, suggesting that human TTR Met 30 binds T4 and retinol-binding protein in vivo. The T4-binding ability of human TTR Met 30 was confirmed by the analysis of T4-binding proteins in the sera of ttr-/- transgenic mice expressing 6.0-hMet 30. The T4-binding studies also demonstrated the presence of hybrid tetramers between mouse and human TTR subunits in the ttr+/+ transgenic mice expressing 6.0-hMet 30.

Although the phenomenon of simultaneous resistance to multiple cytotoxic drugs (multidrug resistance) in cancer cells has been discussed for more than two decades, and the human and mouse genes encoding an energy-dependent transporter (the multidrug transporter or P-glycoprotein) responsible for multidrug resistance were cloned 10 years ago, there is still considerable controversy about the mechanism of action of this efflux pump and its true biological function. This review summarizes the current research on the mechanism of action of the multidrug transporter, including the hydrophobic cleaner and altered partitioning models, the possible function of P-glycoprotein as a chloride and/or ATP channel, the role of phosphorylation in its function and fact and speculation about its physiological role.

Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections. The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo. Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low. In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production. We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES). As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element. In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor). Using a chemiluminescence assay of O2-. production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity. With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2-. production equalling or exceeding that of normal EBV-B cells. These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene. Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.

A procedure for efficient transfer of the human MDR1 (multi-drug resistance) gene into murine hematopoietic stem cells was developed. Cells expressing Sca-1 but no lineage-specific or major histocompatibility complex (MHC) class II antigens (Lin-MHC II-Sca-1+) were enriched from 5-fluorouracil-pretreated bone marrow by Ficoll density-gradient and immunomagnetic sorting. Purified cells were cocultured with growth factors and fibroblasts producing replication-deficient retroviruses containing human MDR1 cDNA. Fluorescence-activated cell sorter analysis and rhodamine-123 efflux experiments showed that greater than 60% of cocultured hematopoietic cells expressed functional human P-glycoprotein. After 6 to 8 days, hematopoietic cells were injected intravenously into sublethally irradiated SCID mice. Stem cell properties of the isolated population were confirmed by sustained expression of MDR1 marker cDNA for greater than 4 to 6 months after transplantation, multilineage engraftment, and presence of MDR1 cDNA in bone marrow of secondary recipient mice after retransplantation. Reconstitution of H-2K-mismatched SCID mice showed high engraftment capacity of Lin-MHC II-Sca-1+ cells. MDR1 cDNA was detected in blood of 78% of recipients. P-glycoprotein was expressed in bone marrow of 71% of mice, in both lymphocytes and myelomonocytoid progenitors. P-glycoprotein function in host marrow was confirmed by rhodamine-123 efflux. Transduction of P-glycoprotein may be useful for gene therapy in two ways: to protect bone marrow from myelosuppression after chemotherapy and as a selectable marker in vivo for the introduction of otherwise nonselectable genes.

Phage HK022 Nun protein excludes phage lambda by terminating transcription near the lambda nut sites. We have established a purified in vitro system that reproduces the in vivo sequence and factor requirements of Nun. Nun arrests transcription by E. coli RNA polymerase at or near elongation pause sites distal to the nut sites. The boxB sequence of nut is required for optimal Nun activity; boxA plays a lesser role. The efficiency of transcription arrest is strongly enhanced by the four E. coli Nus factors. The factors increase the specific activity of Nun, and allow it to act at higher ribonucleoside triphosphate concentrations. A wild-type boxA is required for stimulation by Nus factors. Nun and the lambda N antitermination protein compete for their opposing reactions. This competition may be at the level of binding of boxB RNA.

A temperature-sensitive mutant of DnaK, the principal Escherichia coli member of the 70 kDa heat shock protein family, has been isolated. The mutation, dnaK25, lies in the putative ATP binding pocket of DnaK. It consists of a C to T transition that changes the highly conserved proline 143 to serine. Mutant strains do not support the propagation of bacteriophage lambda or of plasmids that require DnaA for replication. They are also defective in the utilization of mannose and sorbitol. ATPase activity of the mutant protein is reduced 20-fold relative to wild-type, while autophosphorylation is unaffected. DnaK25 has a fourfold faster rate of nucleotide exchange than wild-type DnaK; nucleotide exchange by both proteins is markedly increased by GrpE. The DnaK25 ATPase is still stimulated by DnaJ and GrpE and by peptide substrates. However, the affinity of most peptides tested for stimulating the DnaK25 ATPase is reduced significantly. These results indicate that a mutation in the N-terminal nucleotide binding domain can alter substrate interactions with the C-terminal substrate binding site. Nucleotide exchange by both wild-type DnaK and DnaK25 proceeds at a much faster rate than ATP hydrolysis, and therefore cannot be the rate limiting step of ATP hydrolysis under the conditions used in these experiments. Consistent with this, peptides, which stimulate ATP hydrolysis, have no effect on nucleotide exchange. Peptides thus appear to stimulate the ATPase by acting at another step, such as increasing the rate of phosphate bond cleavage.

A set of heptapeptides was used to compare the relative peptide affinities of three proteins of the hsp70 family: bacterial DnaK, mammalian cytosolic hsc70, and BiP from mammalian ER. Each hsp displays a characteristic pattern of relative affinities. DnaK and hsc70 are more similar to each other than to BiP. A difference in peptide binding specificity may be an important determinant in adjusting an hsp70 family member to its particular cellular function.

The sequence specificity of DnaK substrate binding has been studied using a peptide display library. Based on the amino acid patterns that appeared in this selection, short peptides were synthesized for direct measurements of DnaK affinity. The results show that peptides enriched in internal hydrophobic residues are preferential DnaK substrates, and negatively charged peptides have poor affinity. The isolated C-terminal domain of DnaK binds peptides. Peptide dissociation studies indicate that bound peptides are released from the C-terminal fragment and from DnaK at identical rates. ATP stimulates peptide dissociation from DnaK but not from the C-terminal fragment.

The discovery of an energy-dependent pump system for natural product anticancer drugs has important implications for the biology of related energy-dependent transport systems as well as for the treatment of human cancer. To fully realize the therapeutic potential associated with manipulation of the multidrug transporter, it will be necessary to understand the mechanisms of action of the transporter and its mode of regulation. This review has summarized recent developments in these areas which suggest that both the activity of the pump and its genetic regulation are potential targets for new anticancer therapies.

Gene therapy in humans requires the transplantation of genetically modified cells, and it is important to select only those cells capable of expressing high levels of protein from the transferred gene. Expression of the human multiple drug resistance (MDR) gene confers resistance to a variety of compounds in vitro and in vivo. To determine the feasibility of conferring recipient erythroid cells with the MDR phenotype, we have transduced mouse erythroleukemia cells (MELC) with the MDR gene in a retroviral vector. We show here that MELC clones resistant to exposure to colchicine (an MDR-responsive agent) can be isolated, and demonstrate high levels of MDR RNA and protein expression. Increasing doses of colchicine increase the level of MDR RNA and protein expression significantly. These results indicate that it is possible to transfer and express the human MDR phenotype in mouse erythroid cells by retrovirally mediated gene transfer, and that drug selection can be used to enrich or purify populations of cells containing and expressing this gene.

The lambdoid bacteriophage regulate gene expression by suppressing transcription terminators. Although similar in sequence to lambda, HK022 lacks an analogue to the lambda N antitermination gene and a distinct nutR sequence. To define the HK022 antitermination system, we plated the phage on Escherichia coli nus mutants that inhibit lambda N function. Only rpoB60 (also called nusC60) blocked HK022 lytic growth. Analyses of HK022-lambda hybrid phage suggested that a HK022 function analogous to lambda Q was inhibited by rpoB60. This result was confirmed with pR'-tR'-galK fusions. HK022 Q-protein suppressed tR' in wild-type but not in rpoB60 mutants. The lambda Q-protein, although inhibited by rpoB60, was more active than the HK022 analogue. A single amino acid difference between the two Q-proteins accounts for the phenotype. Changing the penultimate residue of HK022 Q from alanine to the lambda threonine generated a phage that could propagate on rpoB60 hosts. Host and phage mutations that permitted HK022 growth in rpoB60 strains were characterized. The bacterial suppressors were located in the Escherichia coli nusB gene. The phage suppressors represented recessive mutations in a HK022 b-region sequence encoding an open reading frame of 73 codons.

The high level of efficiency of the bacteriophage lambda pL promoter is dependent upon the topological state of the promoter DNA and the binding of a DNA-bending protein, IHF, to a site centered -86 base-pairs upstream from the pL transcription start site. Abortive initiation assays indicate that DNA supercoiling stimulates open complex formation, whereas IHF enhances promoter recognition. IHF stimulates promoter recognition to the same extent on linear and supercoiled templates. We found that the pL region contains a second promoter, pL2, that initiates transcription 42 base-pairs upstream from pL. Although competitive with pL and inhibited by IHF, mutations in pL2 do not affect the regulation of pL. Stimulation by IHF is helix-face-dependent. IHF inhibits pL when the IHF binding site is displaced a helical half-turn upstream. The pL sequences protected against DNase I digestion by bound IHF and RNA polymerase do not overlap. However, DNase I-hypersensitive sites appear in the region between the two bound proteins. In addition, IHF enhances RNA polymerase binding to pL. These data suggest that stimulation of pL by IHF involves the interaction of IHF and RNA polymerase to form a loop or otherwise distort the DNA between their binding sites.

Many human cancers that are initially responsive to chemotherapy eventually fail to respond to treatment. For some drugs, dose escalation that may be required for a cure cannot be achieved because sensitive tissues such as bone marrow (BM) limit cytotoxic therapy. Approaches to prevent or circumvent BM toxicity are therefore a high priority of research on dose escalation protocols. In this study, we have transplanted BM cells from transgenic mice that constitutively express physiologic amounts of a functional human multidrug resistance (MDR1) cDNA to lethally irradiated C57BL/6 x SJL F1 mice (n = 36). From 6 weeks to 10 months after the transplant, all animals contained MDR1 DNA in spleen and BM specimens as indicated by Southern blot analysis, and expressed MDR1 messenger RNA in BM samples as detected by slot blot analysis. In addition, these animals were resistant to the myelosuppressive effect of doxorubicin, daunomycin, taxol, vinblastine, vincristine, etoposide, and actinomycin D, whereas control animals that were reconstituted with normal BM were drug sensitive. Finally, the chemoprotection afforded by the MDR1 gene could readily be reversed by adding chemosensitizers such as cyclosporin A and R-verapamil to chemotherapy. Hence, it appears that BM cells expressing the human MDR1 gene maintain this function after transplantation to host animals for a minimum of 10 months, and confer multidrug resistance to these BM recipients. This selective advantage conferred by expression of the MDR1 cDNA suggests a strategy for the use of MDR1 gene therapy in cancer chemotherapy and for the introduction of otherwise nonselectable genes into BM.

The Escherichia coli rpoB636 mutant is defective in the transcription of lac and other catabolite-sensitive operons. The lac promoter variant, UV5, which is independent of cyclic AMP and the cyclic AMP receptor protein, CRP, was also defective in rpoB636 mutants. The activity of the lac UV5 promoter was restored to wild-type levels by deletion of cya (adenylate cyclase) or crp. Cyclic AMP and CRP apparently act as inhibitors of the rpoB636 RNA polymerase.

The nun gene product of the lambdoid coliphage HK022 provokes premature transcription termination at, or near, the phage lambda nut sites. Termination by Nun and antitermination by lambda N protein both require the nut sites and Escherichia coli NusA, NusB and NusE proteins. To characterize further the host requirements for Nun termination, we selected host mutations that blocked termination at lambda nutR. In addition to mutations in nusA, nusB and nusE, we obtained mutations in rpoC, encoding the RNA polymerase beta' subunit. The nusA and rpoC mutations suppressed Nun termination but not antitermination by lambda N function. The mutations antagonized Nun only at lambda nutR; termination at lambda nutL occurred in all the mutant strains. Thus, nutL is not functionally equivalent to nutR. We conclude that the host requirements for Nun termination overlap but are not identical with those for N antitermination, and, in particular, that the beta' subunit of RNP may be Nun-specific.

Escherichia coli integration host factor (IHF) is a small dimeric protein that binds to a specific DNA consensus sequence and produces DNA bending. Transcription from the bacteriophage lambda pL promoter is stimulated three- to fourfold by IHF both in vivo and in vitro. IHF binds with high-affinity to two tandem sites located just upstream from the pL promoter and enhances the formation of RNA polymerase-promoter closed complexes. The rate of isomerization to open complex is not influenced by IHF. IHF may stimulate recognition of pL by one or more of several mechanisms: (1) by bending DNA; (2) by making protein-protein contacts with RNA polymerase; or (3) by occluding a competing promoter upstream from pL.

The Nun protein of the lambdoid phage HK022 blocks lambda growth by terminating transcription at (or near) the lambda nut sites. An HK022 lysogen carrying a fusion of the lambda pR promoter and nutR site to a gal operon that lacks its own promoter is, therefore, Gal-. To characterize the target of Nun action, spontaneous Gal+ revertants of this strain were isolated and characterized. Two cis-acting mutations are located in the fusion and represent transversions of conserved nucleotides within the boxA sequence (CGCTCTTA) of nutR. One mutation, (CTCTCTTA), is identical with boxA5. The second, boxA16 (CGCTATTA), has not been reported previously. In the absence of Nun, both boxA mutants reduce gal expression. Analysis of in vivo fusion RNA indicates that the mutations increase termination at or near tR1, a rho-dependent lambda terminator located upstream from the fusion point. In contrast to the nutR+ fusion, Nun stimulates gal expression in the boxA mutants by suppressing transcription termination in the tR1 region. Nun antitermination, however, does not extend to distal terminators. The lambda N-function also suppresses termination at or near tR1 in the mutant fusions. N fails to suppress terminators distal to tR1 in the boxA5 fusion, but displays persistent antitermination activity in the boxA16 fusion. A similar reversal of Nun activity occurs when wild-type fusions are introduced into nusA1, nusB5 or nusE71 hosts. We therefore suggest that Nun and N can interact with RNA polymerase in the absence of wild-type boxA, nusA, nusB or nusE, but that the complex formed with mutant components differs functionally from wild-type.

Multidrug-resistance of human cancer cells may result from expression of a 170,000 dalton multidrug efflux pump called P-glycoprotein. To identify this multidrug transporter, and to study its structure and function, we have generated polyclonal rabbit antibodies against the amino-terminal and carboxy-terminal halves of the molecule using recombinant protein fragments produced in Escherichia coli. Two recombinant P-glycoprotein fragments, representing amino acids 140-228 and 919-1280, were overproduced in Escherichia coli by an inducible T7 expression system, gel-purified and injected into rabbits. Both antisera specifically immunoprecipitate 3H-azidopine and 35S-methionine labeled P-glycoprotein from multidrug-resistant cells and detect P-glycoprotein on Western blots with high sensitivity. Because these antisera were raised against epitopes in the amino- and carboxy-terminal halves of P-glycoprotein, they should be useful as research tools to define the function of these two halves of the molecule.

A simple and rapid procedure has been developed to determine similarities in amino acid composition among cellular proteins separated by two-dimensional gel electrophoresis. Cells in tissue culture are simultaneously labeled with two different amino acids each tagged with a different radioisotope. The proteins are then separated on two-dimensional gels and their location on the gels determined by Coomassie-blue staining or autoradiography. Elution of the protein from the appropriate region of the gel followed by liquid scintillation counting yields an isotope ratio which reflects the ratio of the two amino acids in the protein. Examples of the use of this technique in analyzing mutant proteins, proteins altered by carbamylation, and cell proteins with similar amino acid composition (e.g., actin and tubulin) are given.

The immunity region of the lambdoid phage, HK022, has been sequenced. The HK022 repressor gene, its cognate operators and promoters, and several early phage genes can be discerned. The overall design of the immunity region resembles that of other lambdoid phages. The location of the HK022 nun gene, whose product excludes superinfecting lambda by terminating transcription at (or near) the lambda nut sites, is analogous to that of gene N in lambda. nun is preceded by sequences similar to the lambda nut sites and the lambda pL promoter and is followed by several transcription termination signals. Despite these similarities, Nun is required neither for the lytic nor the lysogenic pathway of phage development. Again, unlike N, Nun is expressed in a prophage, perhaps from a promoter other than pL. We suggest that Nun and N have diverged in evolution and now perform different functions for their respective phages. Although Nun and N compete at the lambda nut sites and interact with the same host Nus proteins, they are only distantly related in predicted amino acid sequence. The presence of transcription terminators in the pL operon suggests that the expression of the HK022 early functions, like those of lambda, entails an antitermination mechanism. However, Nun does not appear to be an essential component of this mechanism. Our most economic model is that the HK022 nutL sequence suppresses pL operon terminators in the absence of a phage-encoded antitermination protein. Striking homologies between the HK022 nutL sequence and related sequences in the Escherichia coli rrn operons support this notion. Alternatively, a phage antitermination gene may be located outside the pL operon.

The bacteriophage lambda N protein regulates phage development by selectively suppressing transcription termination in its host, Escherichia coli. The E. coli nus mutants prevent N activity. To provide additional information on transcription termination, we have isolated pseudo-revertants of the nusA1 mutation that restore lambda N function. One series of pseudo-revertants lie in the E. coli nusB gene, whose product is normally required for lambda N activity. These mutations are N-specific: mutations that restore lambda N activity do not restore the activity of the analogous N protein of phage 21. Similarly, nusB mutations that restore phage 21 N function are deficient for lambda N function. Mapping of the two classes of mutation is consistent with their location in two distinct domains in the nusB protein. We discuss whether nusB is specific for N protein or for some other component of this regulation system, e.g. the phage site (nut) required for N action.

The cin-1 mutation creates a new promoter (pcin) in the tR1 region of bacteriophage lambda. The pcin promoter transcribes the cI repressor gene constitutively. lambda cin-1 does not propagate on Escherichia coli mutants lacking the integrative host factor (IHF). lambda cI- cin-1 grows normally in IHF- mutants, indicating that repressor overproduction from pcin blocks lytic growth. The presence of an IHF binding site which overlaps the pcin promoter led us to the hypothesis that IHF functions as a repressor of pcin transcription. We find that the pcin promoter is fivefold more active in a host lacking IHF than in wild-type cells. In vitro studies show that IHF directly inhibits transcription initiation at pcin. Abortive initiation and gel retardation assays demonstrate that IHF interferes with the binding of RNA polymerase to the pcin promoter. RNA polymerase bound in an open promoter complex is resistant to IHF. We propose that IHF binding to the pcin promoter region blocks the binding of RNA polymerase to the promoter, either by covering specific nucleotides or by distorting DNA structure.

The development of simultaneous resistance to multiple drugs in cultured cells occurs after selection for resistance to single agents. This multidrug-resistance phenotype is thought to mimic multidrug-resistance in human tumors treated with chemotherapy. Both the expression of a membrane protein, termed P170 or P-glycoprotein, and the expression of a cloned DNA fragment, termed mdr1, have been shown independently to be associated with multidrug-resistance in cultured cells. In this work, we show that human KB carcinoma cells which express the mdr1 gene also express P-glycoprotein, and that cDNAs encoding P-glycoprotein cross-hybridize with mdr1 cDNAs. Thus, the mdr1 gene codes for P-glycoprotein.

The major excreted protein of transformed mouse cells is an acid activable cysteine protease. In this paper, oxidized insulin B chain is shown to be a substrate for this protease. By isolation and analysis of the insulin B peptides generated by the protease, the bond specificity of this protease was determined. The bonds preferentially cleaved are glu13-ala14, leu17-val18, and tyr26-thr27. No obvious preference for a specific amino acid was found in these studies. The bond specificity of this cysteine protease for oxidized insulin B chain has been compared with that of other proteases, and it is the same as that reported for cathepsin L, suggesting that the major excreted protein and cathepsin L may be the same protein.

We isolated a full-length MDR1 cDNA from human adrenal where P-glycoprotein is expressed at high level. The deduced amino acid sequence shows two amino acid differences from the sequence of P-glycoprotein obtained from colchicine-selected multidrug resistant cultured cells. The amino acid substitution Gly----Val at codon 185 in P-glycoprotein from colchicine resistant cells occurred during selection of cells in colchicine. As previously reported, cells transfected with the MDR1 cDNA carrying Val185 acquire increased resistance to colchicine compared to other drugs. The other amino acid substitution Ser----Ala at codon 893 probably reflects genetic polymorphism. The MDR1 gene, the major member of the P-glycoprotein gene family expressed in human adrenal, is sufficient to confer multidrug-resistance on culture cells.