Proliferative genes induce somatic pairing defects in Drosophila melanogaster and allow replication.

Drosophila tumor forming lines (malignant brain tumor, lethal giant larvae, discs large, brain tumor, and tumor suppressor gene) exhibit incomplete somatic pairing of specific regions in the salivary gland chromosomes, indicating that excessive cell proliferation correlates with somatic pairing defects in Drosophila. Alleles of malignant brain tumor enhancing the frequency of cell divisions exhibit melanizing tumors in the larvae. The giant chromosomes are defective in somatic pairing, indicating that a functional component of the chromosomes is influenced. Genes at different sites are affected, but the similarity of the phenotypes and complex complementation pattern reveals that their functions are interrelated. In the brain of malignant brain tumor recombinants and mutants in proliferative genes, polytene cells appear; wildtype does not amplify DNA in brain tissue cells. Thus, mutant proliferative genes induce the S-phase and allow replication of DNA.

Suppression of c-fos induction in rat brain impairs retention of a brightness discrimination reaction.

Recently, the induction of transcription factor-encoding immediate-early genes such as c-fos was observed in distinct brain regions of rats trained to acquire a footshock-motivated brightness discrimination in a Y-maze. The functional relevance of inducible transcription factors for learning and memory formation is, however, not clear. To address this question in the present study, we have used a synthetic antisense phosphorothioate oligodeoxynucleotide to suppress in vivo the expression of c-fos in rat brain. Intrahippocampal application of the oligodeoxynucleotide 10 hr and 2 hr before starting a brightness discrimination training drastically reduced the induction of c-Fos immunoreactivity normally observed in limbic and cortical areas after the training session. Acquisition of the discrimination reaction was not affected by this treatment. In a relearning test 24 hr after the first training, retention of the discrimination reaction was specifically impaired compared with rats pretreated with control oligodeoxynucleotide or saline. Our findings are consistent with the hypothesis that the inducible transcription factor c-Fos is involved in processes underlying the formation of long-term memory.

Three mutant genes cooperatively induce brain tumor formation in Drosophila malignant brain tumor.

The Drosophila melanogaster strain Malignant Brain Tumor reveals temperature-sensitive transformation of the larval brain tissue. Genetic analysis shows that three gene defects, spzMBT, yetiMBT, and tldMBT, cooperatively induce brain tumor formation. Whereas spz and tld belong to the genes inducing differentiation patterns in the embryo, yeti induces cell overgrowth. spzMBT-, yetiMBT-, and tldMBT-containing animals are larval lethal, whereas Malignant Brain Tumor is kept as a homozygous strain at a permissive temperature. This reveals that this tumor-forming strain is the result of a number of adaptive mutation events.

Receptor specificity of the Escherichia coli T-even type phage Ox2. Mutational alterations in host range mutants.

The T-even type Escherichia coli phage Ox2 uses the outer membrane protein OmpA as a receptor. The protein is recognized with the ends of the virion's long tail fibers. The 266 residue protein 38 is located at this site and acts as an adhesin. Host-range mutants had previously been isolated from Ox2. Mutant Ox2h5 is able to infect cells possessing an altered OmpA protein, which renders the cell resistant to Ox2. Ox2h10 was selected from Ox2h5. This phage recognizes the OmpC protein in addition to the OmpA protein. Ox2h12, which stems from Ox2h10, binds to OmpC with high affinity, but has lost efficient binding to OmpA. The mutational alterations caused in genes 38 are: Asp231----Asn(h5) and His170----Arg(h10). The triple mutant Ox2h12 possesses an insertion of a Gly residue next to Gly121. The three mutants have additionally acquired mutations affecting their base plate, making them "trigger-happy". When protein 38 was compared with the same protein derived from other E. coli phages, it was found to contain two constant and one variable domains, the latter harboring four hypervariable regions flanked by a largely conserved glycine-rich sequence. The h5 and h10 mutations occurred within two hypervariable areas, while the additional Gly residue was present in one of the flanking conserved sequences. On the basis of these results, as well as those obtained from host-range mutants analyzed previously, a model for such adhesins is proposed. Receptor recognition is most likely performed via the hypervariable regions, which may form loops held together in close proximity by the oligoglycine sequences. The latter may achieve this by being part of highly compact omega loops.

Lactose permease of Escherichia coli: properties of mutants defective in substrate translocation.

Mutants of lactose permease of Escherichia coli with amino acid changes (Gly-24----Glu; Gly-24----Arg; Pro-28---Ser; Gly-24, Pro-28----Glu-Ser and Gly-24, Pro-28----Arg-Ser) within a putative membrane-spanning alpha-helix (Phe-Gly-Leu-Phe-Phe-Phe-Phe-Tyr-Phe-Phe-Ile-Met-Gly- Ala-Tyr-Phe-Pro-Phe-Phe-Pro-Ile) are incorporated into the cytoplasmic membrane. The mutant proteins retain the ability to bind galactosides, and the affinity for several substrates is actually increased. However, the rate of active transport is decreased to 0.01% of the wild-type rate in the mutants carrying Arg-24 or Arg-24, Ser-28. Kinetic analysis demonstrates that the two mutants require 10 min to cause occupied binding sites for galactoside and H+ to change their exposure from the periplasm to the cytoplasm as compared to 50 ms in the wild type. The effect is less pronounced when these sites are unoccupied.

Lysis gene t of T-even bacteriophages: evidence that colicins and bacteriophage genes have common ancestors.

The lysis gene t of the T-even-like bacteriophage K3 has been cloned and sequenced. The gene codes for a protein with a predicted molecular weight of 25,200. Expression of the complete lysis protein was impossible, but peptides complementing T4 amber mutants in t are described. No known lysis protein of other phages is homologous to protein T. Also, the Escherichia coli phospholipase A is different from protein T. CelB, the lysis protein of the colicin E2 operon, shows a similarity to protein T. Sequences of colicins A, E1, and E2 are related to gene 38 sequences, the gene preceding t and coding for the phage adhesin. A common origin for colicin genes and phage genes is discussed, and a protein region in colicins that is responsible for receptor recognition is predicted.

Receptor-recognizing proteins of T-even type bacteriophages. Constant and hypervariable regions and an unusual case of evolution.

Proteins 38 of bacteriophages T2, K3, Ox2 and M1 are located at the free ends of their long tail fibers and function as adhesins, i.e. they mediate binding to the bacterial receptors. The latter three phages use the Escherichia coli outer membrane protein OmpA as a receptor, while T2 uses the outer membrane proteins OmpF or Ttr. The DNA sequences of genes 38 of phages Ox2 and M1 have been determined and are compared with those known for T2 and K3. The genes encode 262(T2), 260(K3), 266(Ox2) and 262(M1) amino acid residues. Three domains are distinguishable in these proteins. There are two conserved regions encompassing about 120 NH2-terminal and about 25 CO2H-terminal residues, respectively. The area between these was found to be hypervariable, and it is shown that a very large number of amino acid substitutions, deletions and/or insertions have occurred. Glycine-rich stretches are present within and flanking these areas. Their positions are essentially conserved, indicating an important structural role in receptor recognition. The hypervariability, most likely caused by a constant struggle with bacterial phage-resistant mutants, is so drastic that one cannot discern that T2 uses different receptors from those of the other phages. The partially known sequence of gene 38 of phage T4 has been completed. The gene encodes a protein consisting of 183 amino acid residues. The amino acid composition and sequence of this protein is completely different from those of phages T2, K3, Ox2 and M1. Also, the protein is functionally unrelated to the other proteins 38: it is not present in phage T4 and, unlike the other proteins 38, is required for the efficient dimerization of protein 37. All phages under study are of the same morphology and the genomic organization of the tail fiber genes is identical, with genes 36, 37 and 38 most likely representing, in this order, a transcriptional unit. Sequence similarities between the CO2H-termini of genes 37 of the non-T4 phages and gene 38 of phage T4 were found; this part of gene 37 does not exist in T4. It is suggested that gene 38 of phage T4 originated from a segment of gene 37 of a T2-type phage. Gene 38 of phage T4 is not unique, DNA-DNA hybridization experiments indicated that two other T-even type phages, TuIa and TuIb, possess a T4-type gene 38.

Predicted structure of tail-fiber proteins of T-even type phages.

The sequences of the tail fiber protein 36 of the phages T4, T2, K3, and Ox2 were analyzed for homologies and for folding patterns using structure prediction methods. No repeating motif was found. A model for the fiber structure is proposed in which beta-strands of about 6 amino acids are separated by turns. In the beta-strand, hydrophobic amino acids are found alternating with hydrophilic ones. Such amphipathic beta-strands can be stabilized by dimer formation. The dimerization occurs in a parallel fashion so that both N-termini are at one end of the dimer. This structure represents a rigid fiber. Our model is consistent with electron microscopic data and electron diffraction patterns for the T4 tail fiber. The observation that all fiber components are found as dimers supports our model. Sequences of the receptor recognition proteins 38 of T-even type phages reveal an architecture different from the architecture of the fiber proteins 36 and 37 of these phages.

T-even-type bacteriophages use an adhesin for recognition of cellular receptors.

Protein 38 of the Escherichia coli phage T4 is thought to be required catalytically for the assembly of the long tail fibers of this phage. It is shown that this protein of phage T2 and the T-even-type phage K3 and Ox2 act differently. It was found that NH2-terminal fragments of the protein, expressed from cloned fragments of gene 38 of phage K3, bind to gene 38 amber mutants of phage T2. Such phage or T2 gene 38 amber mutants, grown on a non-permissive host, possess a complete set of six tail fibers but are non-infectious. Both types of non-infectious phage could be repaired by incubation with an extract of cells harboring a cloned gene 38 of a host range mutant of phage K3, K3hx. The repaired phages had the host range of K3hx and not of T2. Immuno-electron microscopy showed that protein 38 is located at the free ends of the long tail fibers of phages T2, K3 and Ox2. The protein serves the recognition of the cellular receptor, i.e. it acts as an adhesin.

DNA sequence of genes 38 encoding a receptor-recognizing protein of bacteriophages T2, K3 and of K3 host range mutants.

Genes 38, which code for a receptor-recognizing protein present at the tip of the long tail fibers, have been sequenced from phages T2, the T-even-type phage K3 and its host range mutants K3hx, K3h1 and K3h1h. The genes from phages T2 and K3 code for proteins consisting of 262 and 260 amino acid residues, respectively. Fifty amino-terminal and 25 carboxy-terminal residues are highly conserved. The amino-terminal amino acids are most likely involved in binding to the neighboring protein 37. Between residues 116 and 226 of the T2 protein and residues 116 and 223 of the K3 protein, sequences exist that are similar to sequences present in Escherichia coli outer membrane proteins and which serve as phage receptors. Most likely, all of these regions in the latter proteins are exposed on the cell surface and are part of their phage receptor areas. In the phage proteins, these sequences are flanked by stretches rich in glycine, perhaps providing an increased flexibility for the polypeptide at these sites; some "wobble" may be required during the protein 38-receptor interaction. The mutational alterations in the host range mutants were found in gene 38. In the K3hx protein, a duplication of six base-pairs caused the wild-type sequence -Gly163-Lys-Leu-Ile- to be changed to -Gly163-Lys-Leu-Lys-Leu-Ile-. In the K3h1 protein, a glutamic acid residue at position 203 was substituted by a lysine. Both alterations occurred within areas similar to outer membrane proteins. Mutant K3h1h, derived from K3h1, exhibits an extended host range as compared to K3h1. No mutational alteration, in addition to that found in K3h1, was found in g38 nor was the part of gene 37 that encodes the carboxy-terminal moiety of the protein altered. K3h1h may represent a "trigger-happy" phage. The results of this and other work show that the phage-phage receptor systems under study represent a primitive immune system.

Receptor specificity of the short tail fibres (gp12) of T-even type Escherichia coli phages.

Short tail fibres of T-even like phages are involved in host recognition. To determine the specificity of the fibres, the region containing gene 12 of phages T2, K3, and K3hx was cloned. The genes 11, 12, wac, and 13, coding for the baseplate outer wedge, short tail fibres, collar wishes, and a head completion component, respectively, were localized on the cloned fragments. Plasmid-encoded gene 12 could be expressed without helper phage. Efficient expression of gene 12 from T2 and K3hx made an extraction of protein 12 possible. Hybrid phages obtained by in vitro complementation, recombination analysis and protein 12 binding to host range mutant bacteria excluded a role of the short tail fibres from T2, K3 or K3hx in the recognition of outer membrane proteins. Binding patterns of protein 12 to different Escherichia coli lipopolysaccharide mutants and inhibition of binding of protein 12 by a monoclonal antibody against the core region of E. coli K12 lipopolysaccharide suggested that heptose residues are necessary for efficient binding. The binding site of the same monoclonal antibody is different from the short tail fibre binding site in an E. coli B strain suggesting different binding specificities of protein 12. Thus, the ability of different bacterial strains to inactivate phage could be related to differences in the binding specificity of the short tail fibres for the lipopolysaccharides of these bacteria.

T-even type phages can change their host range by recombination with gene 34 (tail fibre) or gene 23 (head).

T-even type phages recognize their cellular receptors with the tip of their long tail fibres. The gene products involved in receptor recognition are proteins 37 and 38. While screening libraries of phage K3 with a probe of gene 38 from phage T2, a class of weakly hybridizing clones was found in addition to the expected clones of gene 38 of K3. One of these clones was identified as being from gene 23 of the phage which codes for the major head subunit; another clone originated from gene 34, which codes for the proximal half of the long tail fibres. Neither gene product 23 nor 34 is involved in receptor recognition. Phages can recombine with the DNA of the gene 23 and gene 34 clones and change the host range.

Morphogenesis of the long tail fibers of bacteriophage T2 involves proteolytic processing of the polypeptide (gene product 37) constituting the distal part of the fiber.

Gene 37 of phage T2 codes for a protein that, as a dimer, constitutes the most distal, receptor-recognizing part of its long tail fibers. It was found that, from a plasmid carrying a fragment of gene 37 that lacked a region of the gene encoding 87 CO2H-terminal amino acid residues, a protein was expressed that was slightly larger than that present in the phage. This size difference could not be accounted for. The missing region of gene 37 and also gene 38 (which codes for the auxiliary protein required for dimerization of protein 37) were cloned. Plasmids were constructed with gene 37, or gene 37 together with gene 38, under inducible control. Independent of the presence of the latter gene, a protein was produced that had the same size as protein 37 in the phage. A pulse-chase experiment revealed that a precursor of protein 37 is synthesized and processed such that approximately 120 amino acid residues, most likely CO2H-terminal, are removed. Therefore, the protein produced from the truncated gene was larger because it cannot be processed. This fact also solved an old puzzle: an amber fragment of protein 37 of phage T2 had been found to be larger than the mature protein. The amber codon could be located 24 codons away from the normal stop codon. Obviously, the fragment cannot be processed. The existence of this fragment demonstrates that processing occurs during phage maturation.

Evidence that TraT interacts with OmpA of Escherichia coli.

The OmpA protein is one of the major outer membrane proteins of Escherichia coli. Among other functions the protein serves as a receptor for several phages and increases the efficiency of F-mediated conjugation when present in recipient cells. TraT is an F-factor-coded outer membrane lipoprotein involved in surface exclusion, the mechanism by which E. coli strains carrying F-factors become poor recipients in conjugation. To determine a possible interaction of TraT with OmpA, the influence of TraT on phage binding to cells was measured. Because TraT inhibits inactivation of OmpA-specific phages it is suggested that TraT interacts directly with OmpA. Sequence homology of TraT with proteins 38, the phage proteins recognizing outer membrane proteins, supports this finding. A model of protein interactions is discussed.

DNA sequence of the tail fiber genes 37, encoding the receptor recognizing part of the fiber, of bacteriophages T2 and K3.

The DNA sequences of genes 37 of bacteriophages T2 and K3 are presented and compared with that of phage T4. The corresponding proteins constitute, as dimers, the part of the long tail fibers that recognizes the bacterial receptor. The CO2H termini of the polypeptides are located at the free ends of the fibers. Morphologically, the three phages are essentially identical, but they use different receptors. The genes from phages T4, T2 and K3 encode proteins consisting of 1026, 1341 and 1243 amino acid residues, respectively. DNA-DNA hybridizations had shown earlier that genes 37, in contrast to the gene for the major capsid protein, of a number of T-even type phages are highly polymorphic. The deduced amino acid sequences now show that this polymorphism extends to the protein primary structures. About 50 NH2-terminal residues are conserved and are probably required for binding to the adjacent protein 36. This area is followed by more or less irregularly spaced regions of non-homology, partial homology or complete homology. The heterogeneity is most prominent in a region encompassing about 600 CO2H-terminal residues of the T2 or K3 proteins. Nevertheless, the amino acid compositions of the three proteins are very similar and all are rich in glycine. It has been found that the receptor specificities of phages K3 and T2 are determined by protein 38, a polypeptide required for the efficient dimerization of protein 37 of phage T4. Proteins 38 of phages K3 and T2 are functionally interchangeable, those of T4 and T2 or K3 are not. Proteins 37 of phages K3 and T2 possess a conserved sequence of 160 CO2H-terminal residues. This area is missing in the T4 protein. This region may serve as a binding site for polypeptides 38 of phages K3 and T2. The overall picture of the protein primary structures of the three phages strongly suggests that the evolution of genes 37, which was most likely driven by selection for variations in receptor recognition specificities, has not been a steady process but has involved loss and gain of segments of DNA.

Molecular cloning of the mouse cell adhesion molecule uvomorulin: cDNA contains a B1-related sequence.

A clone (F20) containing coding sequences for the cell adhesion molecule uvomorulin was isolated by immunological techniques from cDNA library in the expression vector lambda gt11. The beta-galactosidase-uvomorulin fusion protein was used to affinity purify anti-uvomorulin antibodies. Affinity-purified antibodies recognized uvomorulin from cell lysates of embryonal carcinoma cells and reacted with the cell surface of embryonal carcinoma cells. The 1.8-kilobase cDNA insert hybridized to a single 4.3-kilobase poly(A)+ RNA species found only in cells expressing uvomorulin. Part of the nontranslated 3' sequences of the cloned uvomorulin cDNA is homologous to the interspersed B1 repeat of the mouse genome.

The receptor specificity of bacteriophages can be determined by a tail fiber modifying protein.

T-Even type bacteriophages recognize their cellular receptors with the distal ends of their long tail fibers. The distal part of these fibers consists of a dimer of gene product (gp) 37. The assembly of this gp to a functional dimer requires the action of two other proteins, gp57 and gp38. Genes (g) 38 have been cloned from five T-even type phages which use the Escherichia coli outer membrane protein OmpA as a receptor. The phages used differ in their ability to infect a series of ompA mutants producing altered OmpA proteins, i.e., each phage has a specific host range for these mutants. The cloned genes 38 complemented g38 amber mutants of phage T2, which uses the outer membrane protein OmpF as a receptor. The complemented phages had become phenotypically OmpA-dependent and, with one exception, OmpF-independent, but regained the host range of T2 upon growth in a host lacking the cloned g38. The host range of the complemented phages, as determined on the ompA mutants, was identical to, similar to, or different from that of the phage, from which the cloned g38 originated. The results presented show that gp38 from one phage can phenotypically 'imprint', in a finely-tuned manner, a host range onto gp37 of another phage with a different host specificity. In view of the extreme diversity of host ranges observed, it is suggested that gp38 of T2 and of the OmpA-specific phages may remain attached to gp37 in the phage particle and in cooperation with gp37 determine the host range.

Presence of DNA, encoding parts of bacteriophage tail fiber genes, in the chromosome of Escherichia coli K-12.

The classical T-even bacteriophages recognize host cells with their long tail fibers. Gene products 35, 36, and 37 constitute the distal moiety of these fibers. The free ends of the tail fibers, which are formed by the CO2H terminus of gene product 37, possess the host range determinants. It was found that 4 out of 10 different strains of Escherichia coli K-12 contained regions of chromosomal DNA which hybridized with a probe consisting of genes 35, 36, and 37 of the T-even phage K3. From one strain this homologous DNA, which was associated with an EcoRI fragment of about 5 kilobases, was cloned into plasmid pUC8. Two independently recovered hybrid plasmids had undergone a peculiar rearrangement which resulted in the loss of about 3 kilobases of cloned DNA and a duplication of both the vector and the remaining chromosomal DNA. The mechanisms causing this duplication-deletion may be related to that of transposases. The cloned DNA was capable of recombination with phage T4 gene 36 and a phage T2 gene 37 amber mutant. DNA sequencing revealed the existence of regions of identity between the cloned DNA and genes 36 and 37 of phage T2. In addition, after growth of a derivative of phage K3 on a strain harboring T2 DNA, it was found that this phage contained the same parts of the T2 tail fiber genes which had been recovered from the bacterial chromosome. There appears to be little doubt that the phage had picked up this DNA from the host. The possibility is considered that a repertoire of parts of genes 36 and 37 of various T-even-type phages is present in their hosts, allowing the former to change their host ranges.

Properties of a mutant lactose carrier of Escherichia coli with a Cys148----Ser148 substitution.

The cysteine residue at position 148 in the lactose carrier protein of Escherichia coli has been replaced by serine using oligonucleotide-directed, site-specific mutagenesis of the lac Y gene. The mutant carrier is incorporated into the cytoplasmic membrane to the same extent as the wild-type carrier, confers a lactose-positive phenotype on cells, and actively transports lactose and other galactosides. However, the maximum rate of transport for several substrates is reduced by a factor of 6-10 while the apparent affinity is reduced by a factor of 2-4. Carrier activity in the mutant is much less sensitive to sulfhydryl reagents (HgCl2, p-(chloromercuri)benzenesulfonate and N-ethylmaleimide) than in the wild type, and beta-D-galactosyl 1-thio-beta-D-galactoside does not protect the mutant carrier against slow inactivation by N-ethylmaleimide. It is concluded that the Cys148 residue is not essential for carrier-catalyzed galactoside: proton symport and that its alkylation presumbly prohibits access of the substrate to the binding site by steric hindrance. A serine residue at position 148 in the amino acid sequence appears to alter the protein structure in such a way that one or more sulfhydryl groups elsewhere in the protein become accessible to alkylating agents thereby inhibiting transport. Recently, Trumble et al. [(1984) Biochem. Biophys. Res. Commun. 119, 860-867] arrived at similar conclusions by investigating a mutant carrier with a Cys148----Gly148 replacement.

The nucleotide sequences of the tail fiber gene 36 of bacteriophage T2 and of genes 36 of the T-even type Escherichia coli phages K3 and Ox2.

Genes 36 have been cloned from phage T2 and the T-even type phages K3 and Ox2. The products of these genes are part of the long tail fibers of the phages, they form the proximal moiety of the distal half fiber. The genes have been sequenced, the nucleotide sequence of gene 36 of phage T4 is known (Oliver, D.B. & Crowther, R.A. (1981) J.Mol.Biol. 153, 545-568). Comparison of the deduced amino acid sequences of the four proteins revealed a surprising pattern. These sequences can be divided into two highly conserved and one very variable region. The former consist of about 60 NH2-terminal and 70 CO2H-terminal residues flanking the variable middle region of about 100 residues. Thus, an identical and unique morphology can be formed by a number of different primary structures. It is proposed that the conserved areas are involved in binding of the proteins to the neighboring products of genes 35 and 37 and that this function has put constraints on the variability of the primary protein structure. The overall amino acid composition of the proteins is rather similar; the codon usage is that known for phage T4. The intercistronic region between genes 35 and 36 consisting of 62 base pairs and containing a presumed terminator for g35 transcription and the 'late type' promoter for transcription of genes 36, 37, and 38, is almost completely identical in the four phages.