Modification enhancement by the restriction alleviation protein (Ral) of bacteriophage lambda.

The product of the lambda ral gene alleviates restriction and enhances modification by the Escherichia coli K-12 restriction and modification system. An open reading frame (orf) located between genes N and Ea10 has been assigned to the ral gene. We have cloned this orf in a plasmid where its transcription is controlled by a thermolabile lambda repressor. Inactivation of the lambda repressor caused a 1000-fold reduction in K-specific restriction of unmodified lambda phage and a 100-fold increase in modification. In minicells transformed with ral+ plasmids, derepression resulted in the appearance of a polypeptide with a lower mobility than that predicted for a protein encoded by the orf attributed to ral; in a transcription and translation system in vitro DNA from a ral+ plasmid encoded a polypeptide with the same mobility. This polypeptide was absent when the plasmid DNA carried a mutant ral gene. The nucleotide sequence of this mutant gene defined two base changes, one of which inactivates the initiation codon of the orf. The K restriction endonuclease, which is also a K-specific methylase, is encoded by three genes designated hsdR, hsdM and hsdS, although the hsdR polypeptide is not essential for the methylase activity. We show that Ral enhances modification in a host strain lacking the entire hsdR gene, and lambda phages carrying the hsdM and S genes modify their own DNA inefficiently in the absence of Ral, despite the fact that derivatives of these phages provide efficient amplification of the K-specific methylase. Our data support a model in which, as a consequence of the interaction of Ral with either the hsdM or the hsdS polypeptide, the conformation of the enzyme is changed and the efficiency of methylation of unmodified target sites is enhanced. It has been postulated that Ral counteracts Rho, but in our experiments Ral did not relieve transcriptional polarity.

Organization and sequence of the hsd genes of Escherichia coli K-12.

The nucleotide sequence of the hsdR and M genes, together with that for hsdS comprises an 8400 base segment spanning the entire hsd region of Escherichia coli K-12. The three hsd genes are transcribed in the same direction, but from two promoters. hsdR and hsdM are separated by 492 base-pairs, whereas the termination codon of hsdM overlaps the initiation codon of hsdS. pres precedes hsdR, and our data indicate a transcription termination signal in the interval between hsdR and pmod, as expected if transcription of hsdM and S is dependent on pmod. Transcription from pres is not influenced by the products of the hsdM and S genes, and the mechanism whereby restriction is prevented when the hsd region is transferred to a modification-deficient cell remains to be elucidated. A segment of the predicted amino acid sequence of the M polypeptide shares homology with a variety of adenine methylases and may identify part of the active site for methylation of specific adenine residues. The R polypeptide shows homology with a variety of ATPases, and pronounced regions of alpha-helical structure are predicted, one of which is amphipathic.

Facilitated engraftment of human hematopoietic cells in severe combined immunodeficient mice following a single injection of Cl2MDP liposomes.

Transplantation of normal and malignant human hematopoietic cells into severe combined immunodeficient (SCID) mice allows for evaluation of long-term growth abilities of these cells and provides a preclinical model for therapeutic interventions. However, large numbers of cells are required for successful engraftment in preirradiated mice due to residual graft resistance, that may be mediated by cells from the mononuclear phagocytic system. Intravenous (i.v.) injection of liposomes containing dichloromethylene diphosphonate (Cl2MDP) may eliminate mouse macrophages in spleen and liver. In this study outgrowth of acute myeloid leukemia (AML) cells and umbilical cord blood (UCB) cells in SCID mice conditioned with a single i.v. injection of Cl2MDP liposomes in addition to sublethal total body irradiation (TBI) was compared to outgrowth of these cells in SCID mice that had received TBI alone. A two- to 10-fold increase in outgrowth of AML cells was observed in four cases of AML. Administration of 10(7) UCB cells reproducibly engrafted SCID mice that had been conditioned with Cl2MDP liposomes and TBI, whereas human cells were not detected in mice conditioned with TBI alone. As few as 2 x 10(4) purified CD34+ UCB cells engrafted in all mice treated with Cl2MDP liposomes. In SCID mice treated with macrophage depletion unexpected graft failures were not observed. Histological examination of the spleen showed that TBI and Cl2MDP liposomes i.v. resulted in a transient elimination of all macrophage subsets in the spleen, whereas TBI had a minor effect. Cl2MDP liposomes were easy to use and their application was not associated with appreciable side-effects. Cl2MDP liposome pretreatment in combination with TBI allows for reproducible outgrowth of high numbers of human hematopoietic cells in SCID mice.

Lambda Charon vectors (Ch32, 33, 34 and 35) adapted for DNA cloning in recombination-deficient hosts.

New phage lambda-based cloning vectors, Charons 32, 33, 34 and 35, have been constructed. These vectors allow cloning of large (19-21 kb) DNA fragments in up to six cloning sites. DNA cloned in these vectors can be propagated on recA- Escherichia coli hosts.

A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes.

Lambda derivatives are described that can be used for cloning DNA fragments of about 20 kilobase pairs (kb) generated by restriction enzymes EcoRi, HindIII, BamHI, MboI and BglII. Recombinants can be selected by their Spi- phenotype and their propagation is facilitated by the presence of a chi site.

Cloning of the MCF1233 murine leukemia virus and identification of sequences involved in viral tropism, oncogenicity and T cell epitope formation.

MCF1233 is an oncogenic C57BL-derived retrovirus of the Murine Leukemia Virus (MuLV) family, that causes T and B lymphomas in an MHC-associated fashion. In this study, we cloned MCF1233, determined its nucleotide sequence and, by comparison with its MuLV relatives, identified the sequences that relate to the leukemogenic character of this virus. MCF1233 was found to have an ecotropic backbone, and carried acquired polytropic sequences in the 3' pol and 5' env region. The gag-region contained six specific nucleotides, determining the viral B-tropism. Short sequences within the U3 LTR shared specific homology with the xenotropic Bxv-1 MuLV, which is the U3 donor for leukemogenic MCF MuLV of AKR origin. These sequences, in combination with specific ecotropic sequences present in env p15E, most likely determine the viral oncogenicity. Currently, the deduced MCF1233 amino sequence is being exploited for T cell epitope analysis, which in this paper is discussed with respect to antigenically distinct Friend/Moloney/Rauscher types of MuLV. Identification of these T cell epitopes will contribute to our understanding of the fundamental aspects of immune control on MCF1233-induced lymphomagenesis. It will help to elucidate the mechanisms that underlie immune escape of T lymphomas, rarely arising in immunoresistant mice, and allow the development of vaccination protocols for tumor therapy.

Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli.

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.

S-adenosylmethionine: jack of all trades and master of everything?

SAM (S-adenosylmethionine, also known as AdoMet) is well known as the methyl donor for the majority of methyltransferases that modify DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modelling, epigenetic modifications and imprinting, which are all topics of great interest and importance in cancer research and aging. In total, 15 superfamilies of SAM-binding proteins have been identified, with many additional functions varying from methylation of phospholipids and small molecules such as arsenic to synthesis of polyamines or radical formation. SAM is regenerated from demethylated SAM via the methionine cycle, which involves folate. Imbalance of this cycle in humans, e.g. through folate shortage via dietary insufficiency, alcohol abuse, arsenic poisoning or hereditary factors, leads to depletion of SAM and human disease. In addition to its role as a methyl donor to modification enzymes that protect bacterial DNA against cognate restriction, SAM also serves as a co-factor for nucleases such as the type I restriction enzyme EcoKI, which is unable to restrict DNA in the absence of SAM. Finally, on a completely different tack, SAM can bind to certain RNA structures called riboswitches that control transcription or translation. In this way, expression of multiple genes can be regulated in a SAM-dependent manner, an unexpected finding that opens up new avenues into gene control. This minireview discusses some of these diverse and amazing roles of this small metabolite.

CD27 and (TNFR) relatives in the immune system: their role in health and disease.

Relatives of the tumor necrosis factor receptor family are proving of crucial importance for an effective immune response as well as maintenance of homeostasis. The reviews in this issue summarise accumulating evidence for an emerging intricate interplay between these receptors themselves and with the CD28 pathway. It is hypothesised, that the lesser known CD27 receptor may overlap and synergise with CD40 and other relatives and may regulate the T-cell mediated activation cascade and control lymphocyte expansion by facilitating maturation and cell cycle progression. The diagnostic and prognostic value of the occurrence of these receptors and their soluble forms is becoming increasingly apparent, and recent data may herald new strategies for T cell manipulation via combinations of these receptors to ameliorate or prevent immune disease, coronary heart disease, transplant rejection, graft-versus-host disease, viral infections, and to promote tumor eradication.

Immune evasion by human cytomegalovirus: lessons in immunology and cell biology.

The human cytomegalovirus (HCMV) has dedicated a significant part of its genome to genes encoding molecules that modulate the host immune response. Many of these genes have homologues in the host genome. Others, however, are unique in the sense that no obvious primary sequence identity is found in the available databases. The HCMV gene products interfere with the activation of MHC class I and class II restricted T cells and NK cells, modify the function of cytokines and their receptors, interact with complement factors and modulate signal transduction and transcription factor activity, in addition to interference with many other cellular functions. Investigation of these evasion strategies has not only improved our understanding of HCMV pathogenesis, but has also provided unexpected, novel insights into basic cell biological and immunological processes.

A general method for the transfer of plasmid-borne mutant alleles to the chromosome of Klebsiella pneumoniae using bacteriophage lambda: transfer of pqq genes.

A generally applicable method is described for reintroduction of mutant plasmid-borne alleles to the chromosome of Klebsiella pneumoniae using bacteriophage lambda. We used this method to make stable chromosomal transposon insertions in genes for biosynthesis of pyrroloquinoline quinone in K. pneumoniae.

The T cell activation antigen CD27 is a member of the nerve growth factor/tumor necrosis factor receptor gene family.

CD27 is a dimeric membrane glycoprotein found on the surface of most human T lymphocytes. Activation of T cells by engagement of the Ag receptor increases CD27 surface expression, and anti-CD27 antibodies augment Ag receptor-mediated T cell proliferation. In this study a cDNA-encoding CD27 was isolated by expression and immunoselection in COS cells. The predicted polypeptide was found to belong to a recently characterized family of cysteine-rich receptors whose known ligands include nerve growth factor and TNF-alpha and -beta. Structural similarities suggest that CD27 belongs to a lymphocyte-specific subgroup of the family, comprised of the B cell Ag CD40, the rat T cell subset Ag OX40, and the mouse T cell activation Ag 4-1BB. Recent studies suggest some of these molecules may play a role in the survival of activated cells.

The CD27 membrane receptor, a lymphocyte-specific member of the nerve growth factor receptor family, gives rise to a soluble form by protein processing that does not involve receptor endocytosis.

CD27 is a transmembrane glycoprotein found exclusively on human T and B lymphocytes. It belongs to a recently identified receptor family, whose members are involved in cell differentiation and survival. This family includes the nerve growth factor receptor, two different types of tumor necrosis factor, receptors the Fas antigen, and the B cell-specific protein CD40. T cell activation via the antigen receptor strongly enhances CD27 membrane expression, suggesting a role for CD27 during T cell differentiation. A soluble form of CD27 (sCD27) is released into the supernatant of activated T cells, and detected in serum and urine of healthy individuals and patients. We have investigated the mechanism underlying the generation of sCD27. One mRNA encodes both the transmembrane receptor and sCD27, as shown by cDNA transfection. In line with this, only one CD27 precursor protein is found, that is processed to the mature receptor by extensive O-linked glycosylation. All newly synthesized protein is rapidly transported to the plasma membrane; no internal pool of mature protein is detectable. The transmembrane form gives rise to sCD27 after arrival at the cell surface, most likely via a proteolytic event, that does not involve receptor internalization.

Genomic organization and chromosomal localization of the human CD27 gene.

CD27 is a lymphocyte-specific member of a recently identified receptor family with at least 10 members that includes the receptors for nerve growth factor and TNF, CD40, and Fas. Several members of this family play a role in cell differentiation, proliferation, and survival. Within the amino terminal ligand binding domain of these receptors, repeat motifs have been identified. These repeats contain many cysteine residues in a conserved pattern, characteristic of this family. We have isolated and characterized the human CD27 gene to gain insight into the evolution of this type of receptor domain. The gene was localized on chromosome 12, band 12p13. Sequence analysis showed no correlation between the intron/exon organization and the subdivision of the protein into distinct domains. Structural information for the cysteine-rich domain is contained within three exons. In addition, the splice sites in the CD27 gene are located in a different position from those in the related nerve growth factor receptor gene. However, a comparison of the splice sites within the regions encoding the respective ligand-binding domains of the CD27 and nerve growth factor receptor genes identifies the archetypal cysteine-rich building blocks, from which the members of this family may have arisen during the course of evolution. From this observation, we propose a new organization of the repeat motifs.

Molecular characterization of the melanocyte lineage-specific antigen gp100.

The glycoproteins recognized by monoclonal antibody (mAb) NKI-beteb are among the best diagnostic markers for human melanoma because their expression is restricted to melanocytic cells. Recently, we isolated a cDNA clone, termed gp100-c1, which confers immunoreactivity not only to mAb NKI-beteb, but also to two other mAbs used to diagnose malignant melanoma, HMB-50 and HMB-45. In this report, we demonstrate that gp100-c1 cDNA encodes glycoproteins of 100 kDa (gp100) and 10 kDa (gp10) which are recognized by these mAbs in human melanoma cells. The translation product deduced from the open reading frame present in gp100-c1 cDNA is highly homologous to another melanocyte-specific protein, Pmel17. Nucleotide sequence analysis of genomic DNA indicates that the transcripts corresponding to gp100 and Pmel17 cDNAs originate from a single gene via alternative splicing. In all normal and malignant melanocytic cells analyzed, gp100 and Pmel17 RNAs are simultaneously expressed.

Regulation of expression of CD27, a T cell-specific member of a novel family of membrane receptors.

CD27 belongs to a newly defined family of transmembrane R, including the nerve growth factor R, two distinct TNF R and CD40. The function of CD27 is unknown, but on the basis of structural and functional properties, we postulate that it plays a role in the events subsequent to T cell activation, possibly as a cytokine R. We have analyzed the mechanisms underlying the regulation of CD27 protein expression. Membrane expression of CD27 strongly increases after T cell activation via the TCR/CD3 complex or the CD2 molecule. In contrast, direct stimulation of protein kinase C by phorbol esters markedly down-regulates CD27 surface expression. This down-regulation most likely does not result from CD27 phosphorylation, because both anti-CD3 mAb and PMA induce hyperphosphorylation of CD27 on serine residues. Rather, membrane expression seems to be regulated primarily at the RNA level. Stimulation of T cells with anti-CD3 mAb strongly increases steady state CD27 mRNA levels, whereas PMA treatment greatly reduces these transcript levels. Dissection of the TCR/CD3-induced signaling pathways showed that cytoplasmic cAMP as well as Ca2+ concentrations contribute to the increase of CD27 expression. These data indicate that upon Ag-specific T cell stimulation, membrane expression of CD27 is regulated at the RNA level through the joint action of distinct TCR/CD3-associated signaling pathways.

Cloning and expression of murine CD27: comparison with 4-1BB, another lymphocyte-specific member of the nerve growth factor receptor family.

CD27 is a member of the nerve growth factor receptor family, that includes two types of tumor necrosis factor receptor, CD40 and Fas/Apo-1. Human CD27 has been found only on lymphocytes. In T cells, its expression strongly increases in a transient fashion upon antigenic stimulation, suggesting that CD27 plays a role during T cell activation. To analyze the function of CD27, we have identified the murine CD27 at the cDNA and protein level. Murine CD27 shows an identity of 65% compared with human CD27. The amino-terminal cysteine-rich region, i.e. the putative ligand-binding domain, and the carboxy-terminal part of the cytoplasmic domain are approximately 80% identical in man and mouse. Murine CD27 has 29% identity to 4-1BB, another lymphocyte-specific member of the receptor family defined only at the cDNA level. Murine CD27 and 4-1BB have 39% homology in the cysteine-rich domain and share a conserved region in the cytoplasmic tail. Expression studies identified murine CD27 mRNA in thymus and spleen, but not in non-lymphoid tissues, while 4-1BB mRNA was not detected in any tissue tested. In resting T cells, only murine CD27 mRNA was found, while in activated T cells murine CD27 as well as 4-1BB were present at high levels. Murine CD27 and 4-1BB mRNA are expressed with different kinetics during T cell activation, suggesting that these molecules play different roles in this process. Peptide antisera identified murine CD27 as a 45-kDa protein on thymocytes and activated T cells, while 4-1BB was precipitated as a 35-40-kDa protein from activated T cells.