MIP1 alpha nuclear protein (MNP), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human MIP-1 alpha gene.

Murine macrophage inflammatory protein 1 alpha (MIP-1 alpha) and its human equivalent (GOS19, LD78, or AT464) are members of the -C-C family of low-molecular-weight chemokines. Secreted from activated T cells and macrophages, bone marrow-derived MIP-1 alpha/GOS19 inhibits primitive hematopoietic stem cells and appears to be involved in the homeostatic control of stem cell proliferation. It also induces chemotaxis and inflammatory responses in mature cell types. Therefore, it is important to understand the mechanisms which control the expression of MIP-1 alpha/GOS19. Previous work has shown that in Jurkat T cells, a set of widely expressed transcription factors (the ICK-1 family) affect the GOS19 promoter. One member, ICK-1A, behaves as a strong negative regulator. In this communication, we provide evidence that the pathway of induction in the macrophage cell line U937 is different from that in Jurkat cells. Furthermore, we show that the ICK-1 binding site does not confer negative regulation in U937 cells. We provide evidence for an additional binding site, the MIP-1 alpha nuclear protein (MNP) site, which overlaps the ICK-1 site. Interaction of nuclear extracts from various cell lines and tissue with the MNP site leads to the formation of fast-migrating protein-DNA complexes with similar but distinct electrophoretic mobilities. A mutation of the MNP site which does not abrogate ICK-1 binding inactivates the GOS19.1 promoter in U937 cells and reduces its activity by fourfold in Jurkat cells. We propose that the MNP protein(s) binding at the MNP site constitutes a novel transcription factor(s) expressed in hematopoietic cells.

Detection of RNA complementary to herpes simplex virus DNA in human cervical squamous cell neoplasms.

Nonneoplastic and neoplastic cervical biopsy specimens were examined by in situ hybridization to 125I-labeled DNA of herpes simplex virus (HSV), adenovirus, and bacteriophage lambda DNA's, and quantitative hybridization data were obtained using a Video Image Analyser. HSV-specific RNA was detected in 72% of cervical intraepithelial neoplasia, 60% of squamous cervical carcinomas, 2% of nonneoplastic cervices, and 9% of primary adenocarcinomas of the cervix. None of the tissues gave positive hybridization with adenovirus or lambda DNA probes. In paired biopsies of cervical intraepithelial neoplasia and nonneoplastic epithelium from 29 individuals, HSV-specific RNA was detected only in the epithelium of the neoplastic sample and not in the nonneoplastic control. Infectious HSV-2 was isolated from a low proportion (2%) of both ectocervical swabs and cell-free tissue extracts of patients examined, suggesting that the HSV-specific RNA detected in squamous cell neoplasms was not due to overt infections.

Regulatory domains within the P0 promoter of human c-myc.

Expression of P0 RNA in some Burkitt lymphoma cell lines varies independently of levels of RNA derived from P1 and P2. These data suggest the possibility that expression of P0 RNA may be capable of independent regulation. In order to investigate this possibility we have isolated putative regulatory domains flanking P0 RNA starts within the human c-myc gene and analysed both their ability to direct expression of control reporter genes and their ability to interact with specific transcription factors. Regulatory regions necessary for expression of P0 RNA have been located within 131 bp 5' of the first major P0 RNA start. DNAase 1 footprint analysis and gel retardation assays demonstrate binding of transcription factors Sp1, NF1 and CBP to this region. NF1 binds specifically to two consensus sequences. The more distal site overlaps with the binding site for CBP, and it is likely that concomitant binding of NF1 and CBP within the distal region of the P0 promoter is not possible. Previous work from our laboratory has described a negative regulatory domain within the 5' flanking region of c-myc. The P0 promoter resides within this domain and therefore may contain a negative regulator of c-myc gene expression.

Regulation of AP-1/DNA complex formation in vitro.

The level of AP-1 DNA-binding activity exhibited in vitro by unfractionated extracts of Hela nuclei can be stimulated by a low molecular weight fraction from rabbit reticulocyte lysate. Stimulation also requires a heat labile component of the nuclear extract, probably a protein. Stimulated and unstimulated extracts with high and low AP-1 DNA-binding activities contain the same levels of proteins reactive with antisera against Jun and Fos, proteins which are shown to be involved in the AP-1/DNA complexes detected in vitro. The low molecular weight fraction from reticulocyte lysate can be substituted by the reducing agent dithiothreitol (DTT) in the stimulation reaction and conversely oxidised glutathione greatly reduces formation of AP-1/DNA complexes. The binding activities of transcription factors SP-1, NF-1 and CBP are unaffected by DTT or oxidised glutathione. These observations, taken together, suggest that the efficiency with which pre-existing Fos and Jun proteins can bind an AP-1 target sequence in vitro can be controlled by a nuclear activity which is sensitive to oxidation/reduction and that this control mechanism is specific for AP-1.

Phorbol ester-responsive H-ras1 gene promoter contains multiple TPA-inducible/AP-1-binding consensus sequence elements.

We have constructed recombinant DNA plasmids which carry both the aminoglycoside phosphotransferase (aph) gene and the chloramphenicol acetyl-transferase (CAT) gene linked to the human normal or mutant T24 H-ras1 promoter. We have transfected these plasmids into rat 208F fibroblasts using the calcium phosphate technique and selected for stable transformants by geneticin resistance. These transformants expressed CAT activity at low levels. However, when treated with the phorbol ester TPA, CAT levels increased substantially. Cells transfected with recombinant plasmids carrying a promoterless CAT gene did not respond to TPA. We have noted four motifs in the H-ras1 promoter region which resemble TPA-inducible and AP-1-binding consensus sequences. We suggest that AP-1-like proteins may play a role in control of H-ras1 transcription.

The FGF-4 promoter is required for transformation and is active in both embryonal and somatic cells.

We report the independent isolation of a rearranged FGF-4 gene from a patient with chronic myeloid leukaemia. We show that the FGF-4 gene has been truncated 30 nucleotides 3' to the coding sequence and has been fused to the RNA processing signals from a putative unknown gene on chromosome 15. We demonstrate that the promoter region of the FGF-4 gene is active in NIH3T3 cells and is indeed necessary for transformation. Using the luciferase reporter assay we have shown that the FGF-4 5' flanking sequences possess easily detectable promoter activity in both F9 and HeLa cell lines. 5' deletion analysis of the FGF-4 promoter has delineated regions containing cis-acting elements of functional importance. These regulatory regions are common to both embryonal and somatic cell lines. Electrophoretic mobility shift assay, using nuclear extracts from F9 and HeLa cells, has allowed detection of DNA-protein interactions occurring in the functionally significant regions. Subsequent comparison of the human and murine FGF-4 promoters show that the regions of functional significance are highly conserved. We suggest that the FGF-4 gene may be suppressed through a distal suppressor locus and becomes active when separated from this suppressor.

Heterogeneity of cell lines derived after transformation of early passage rodent cells by the Ha-ras1 human oncogene.

The chromosome patterns of Chinese hamster cell lines derived after immortalization or tumorigenic conversion of early passage cells with recombinants carrying the mutated T24 or the normal human Ha-ras1 gene have been characterized by trypsin-Giemsa banding. Whereas immortalized Chinese hamster cell lines exhibited a near normal karyotype, tumorigenic cell lines were found to have abnormal karyotypes carrying marker chromosomes. Moreover, chromosomal patterns correlated with growth in semisolid media and tumourigenicity in nude mice. Similarly, malignant conversion of early passage Syrian hamster cells, with a recombinant carrying the mutated T24 human Ha-ras1 gene, resulted in cells with a near diploid karyotype. On the other hand, tumorigenic conversion of early passage Wistar rat cells with the same oncogene produced cell lines with heteroploid karyotypes. More chromosomal alterations have been observed during further growth of these cells. It is suggested that the transformed phenotype in these cells may be dependent on the chromosomal instability.

Expression of the normal H-ras1 gene can suppress the transformed and tumorigenic phenotypes induced by mutant ras genes.

The transformed phenotype of rat 208F cells transfected with the T24 H-ras1 oncogene is suppressed by simultaneous or subsequent transfection with the normal H-ras1 gene. The suppressed cells express both the normal and mutant forms of ras p21 but the normal form predominates. Rare transformed cells obtained after simultaneous transfection express mainly the T24 p21. Some suppressed cells induce tumours in nude mice after a long lag period and these tumour cell lines have much reduced expression of normal p21. The normal H-ras1 gene also suppresses the transformed phenotype induced by mutant N-ras, albeit less effectively. The tumorigenicity of the EJ bladder carcinoma cell line, which contains only the T24 mutant allele of H-ras1, is also suppressed following transfection with the normal H-ras1 gene. The results suggest that transforming alleles of ras genes do not behave in a fully dominant manner and that expression of the normal allele at elevated levels can lead to suppression of the transformed and tumorigenic phenotypes.

Physical maps for Herpes simplex virus type 1 DNA for restriction endonucleases Hind III, Hpa-1, and X. bad.

It has been proposed that the genome of herpes simplex virus type 1 (HSV-1) consists of two internal unique sequences, S and L, bounded by two sets of redundant sequences (P. Sheldrick and N. Berthelot, 1974). In this arrangement, terminal sequences (TRs and TRl) are repeated in an internal inverted form (IRs and IRl) and delimit S and L. Furthermore, a body of evidence has accumulated that suggests that S and L themselves are inverted, giving rise to four related forms of the HSV genome. In this study the ordering of restruction endonuclease fragments of HSV-1 DNA for physical maps has been studied using molecular hybridization techniques and the cleavage of isolated restriction endonuclease fragments with further restriction endonucleases. Physical maps for the fragments produced by Hind III, Hpa-1, and X. bad have been constructed for the four related forms of the HSV-1 genome. TRs and IRs were found to be between 3.5 x 10(6) and 4.5 x 10(6) daltons, TRl and IRl about 6 x 10(6) daltons, S about 8 x 10(6) to 9 x 10(6) daltons, and L about 6.8 x 10(6) daltons.

The normal human H-ras1 gene can act as an onco-suppressor.

The altered morphology and tumorigenic phenotypes of rat 208F fibroblasts transformed with the human T24 H-ras1 oncogene is suppressed by transfection with the human normal H-ras1 gene. In the suppressed cells, both the normal and mutant T24 ras gene products are expressed although the normal p21 is expressed at a higher level. Rare transformants or tumours derived from suppressed cells possess reduced expression of normal ras p21. Our findings suggest that transforming ras alleles do not behave in a dominant manner and that elevated expression of the normal allele could cause suppression of the morphologically transformed and tumorigenic phenotypes.

An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA.

Cells infected with herpes simplex virus type 1 (HSV-1) DNA by the calcium phosphate precipitation technique produce virus which leads to the formation of plaques (Graham, Veldhuisen & Wilkie, 1973). In the study reported here we show that treatment of cell monolayers with dimethyl sulphoxide (DMSO) solutions after infection with DNA-calcium phosphate complexes leads to a considerable increase in the number of plaques obtained. The conditions for this enhancement of infectivity have been optimized for baby hamster kidney (BHK) cells, and increases in plaque numbers of over 100-fold have been obtained. The treatment appears to increase the proportion of cells which respond to DNA infection by initiating plaque formation, and results in a large increase in the measured specific infectivity of HSV-1 DNA. DMSO causes similar (but quantitatively different) responses in various other cell lines infected with HSV-1 DNA. BHK cells infected with either virus particles, or virus DNA by the DEAE-dextran technique (Laithier & Sheldrick, 1975), do not exhibit this massive enhancement following exposure to DMSO.

Sequence arrangement in herpes simplex virus type 1 DNA: identification of terminal fragments in restriction endonuclease digests and evidence for inversions in redundant and unique sequences.

It has been proposed by Sheldrick and Berthelot (1974) that the terminal sequences of herpes simplex virus type 1 (HSV-1) DNA are repeated in an internal inverted form and that the inverted redundant sequences delimit and separate two unique sequences, S and L. In this study the sequence arrangement in HSV-1 DNA has been investigated with restriction endonuclease cleavage, end-labeling studies, and molecular hybridization experiments. The terminal fragments in digests with restriction endonucleases Hind III, Hpa-1, EcoRI and Bum were identified and shown to be consistent with the Sheldrick and Berthelot model. Inverted fragments which contain unique sequences as well as redundant sequences, and which the model predicts, were identified by DNA-DNA hybridization studies. Further cleavage of Bum fragments with Hpa-1 also revealed inversions of the terminal sequences that contained unique sequences. The results obtained showed that the unique sequences S and L are relatively inverted in different DNA molecules in the population, resulting in the presence of four related genomes with rearranged sequences in apparently equal amounts. The redundant sequences bounding S do not share complete sequence homology with those bounding L, but hybridization studies are presented which show that the terminal 0.3% of the genome is repeated in every redundant sequence.

Polyribonucleotide synthesis by subfractions of microsomes from rat liver.

1. The microsome fraction of rat liver has been fractionated and the ability of the fractions to incorporate ribonucleotides into polyribonucleotides has been studied. Activity was found in the rough-surfaced vesicle (light) fraction and in the free-ribosome fraction and this latter activity has been examined. 2. The free-ribosome fraction contains ribosome monomers, dimers and trimers together with some higher oligomers and ferritin. In addition to catalysing the incorporation of ribonucleotides into acid-insoluble material it contains diesterase activity. It catalyses the incorporation of UMP from UTP, but not UDP, AMP from ATP and CMP from CTP into polyribonucleotide material, and for UTP the product appears to be a homopolymer not more than eight units long attached to the ends of primer polyribonucleotide strands. 3. The activity could not be removed from the free-ribosome fraction by washing or by isolation in the presence of ethylenediaminetetra-acetic acid. 4. Partially hydrolysed polyuridylic acid but not polyadenylic acid could serve as a primer for the incorporation of UMP, but some activity was always associated with an endogenous primer. 5. Analysis of RNA extracted from the free-ribosome fraction after incubation with [(3)H]UTP showed the presence of 28s, 18s, 5s and transfer RNA types, but no radioactivity was associated with any of these RNA fractions.

Chain extension of ribonucleic acid by enzymes from rat liver cytoplasm.

1. The 105000g supernatant fraction of rat liver catalyses the incorporation of ribonucleotides from ribonucleoside triphosphates into polyribonucleotide material. The reaction requires Mg(2+) ions and is enhanced by the addition of an ATP-generating system and RNA, ATP, UTP and CTP but not GTP are utilized in this reaction. In the case of UTP, the product is predominantly a homopolymer containing 2-3 uridine residues, and there is evidence that these may be added to the 3'-hydroxyl ends of RNA or oligoribonucleotide primers. 2. The microsome fraction of rat liver incorporates ribonucleotides from ATP, GTP, CTP and UTP into polyribonucleotide material. This reaction requires Mg(2+) ions and is enhanced slightly by the addition of an ATP-generating system, and by RNA but not DNA. Supplementation of the reaction mixture with the three complementary ribonucleoside 5'-triphosphates greatly increases the utilization of a single labelled ribonucleoside 5'-triphosphate. The optimum pH is in the range 7.0-8.5, and the reaction is strongly inhibited by inorganic pyrophosphate and to a much smaller degree by inorganic orthophosphate. It is not inhibited by actinomycin D or by deoxyribonuclease. In experiments with [(32)P]UTP in the absence of ATP, GTP and CTP, 80-90% of (32)P was recovered in UMP-2' or -3' after alkaline hydrolysis of the reaction product. When the reaction mixture was supplemented with ATP, GTP and CTP, however, about 40% of the (32)P was recovered in nucleotides other than UMP-2' or -3'. Although the reactions seem to lead predominantly to the synthesis of homopolymers, the possibility of some formation of some heteropolymer is not completely excluded.

Location and orientation of homologous sequences in the genomes of five herpesviruses.

Molecular hybridization experiments were carried out to investigate homologous regions in the genomes of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), equid herpesvirus 1 (EHV-1), pseudorabies virus (PRV) and varicella-zoster virus (VZV). Virion DNA probes from EHV-1, PRV and VZV hybridized to similar regions of the HSV genome, and the use of cloned DNA probes allowed heterologous genomes to be oriented with respect to homologous regions. The HSV-1 and HSV-2 genomes are colinear, the EHV-1 and VZV genomes are colinear with the IL or ISL genome arrangement of HSV, and the PRV genome is essentially colinear with the IL genome arrangement of HSV except that the region 0.1 to 0.4 fractional genome units appears to be inverted. A detailed analysis of sequences in the HSV-2 and PRV genomes to which the HSV-1 major capsid protein gene hybridized was carried out in order to demonstrate the application of molecular hybridization to the location of genes in heterologous genomes. The lesion in a DNA-positive temperature-sensitive mutant of PRV was mapped within the putative PRV major capsid protein gene. We conclude that the herpesviruses we have studied possess several highly conserved genes, and propose that they are similar in genetic organization despite presumably separate evolutionary histories.

Inversion of the two segments of the herpes simplex virus genome in intertypic recombinants.

We have analysed by restriction site mapping the structures of the termini and L-S joint in several HSV-1/HSV-2 intertypic recombinants, including Bx1(28-1), the virion DNA of which has a marked overabundance of one orientation of the L segment, and subclones of Bx1(28-1). All recombinants with both orientations of L present in equal amounts contain TRL and IRL regions derived at least in part from the same parent (HSV-1 or HSV-2) as a result of previously undetected crossovers in these regions. Recombinants with a predominance of one orientation of L have TRL and IRL regions derived from different parents. Homology between a sequences alone at the L terminus and L-S joint is sufficient for normal inversion of L. Analysis of another recombinant, RE4, which fails to invert normally in both L and S, suggests that normal inversion of S is dependent upon the presence of TRS and IRS regions derived at least in part from the same parent. We conclude that segment inversion specifically depends upon the a sequence, that the process of DNA replication and maturation does not necessarily produce molecules with identical a sequences, and that direct ligation of termini may occur during DNA replication.

Either orientation of the L segment of the herpes simplex virus genome may participate in the production of viable intertypic recombinants.

We have analysed the genome structures of 90 recombinant viruses produced by co-infection of baby hamster kidney cells with the DNA of a herpes simplex virus type 1 temperature-sensitive mutant (tsD) and specific DNA fragments of wild-type herpes simplex virus type 2 at non-permissive temperature. Crossovers were located predominantly in regions of greatest intertypic homology, and we conclude that primary recombination occurred with the L segment of the genome in either orientation.

Nucleotide sequences of the joint between the L and S segments of herpes simplex virus types 1 and 2.

The a sequence of herpes simplex virus (HSV) is present as a direct repeat at the genomic termini and also in inverted orientation at the joint between the L and S segments. DNA sequences have been determined for the joint regions of the genomes of HSV-1 and HSV-2, and relative to these sequences the genomic termini are in both cases located close to a short direct repeat of 17 to 21 base pairs (bp) at the b-a and a-c junctions. The HSV-1 joint region contains three separate tandem direct reiterations of short sequences, (12, 16 and 17 bp in strain 17) and we conclude that size heterogeneity in the a and c sequences is due to variable copy numbers of these repeated units. It is likely that a considerable part of the HSV-1 joint region does not code for polypeptide.

Physical maps for HSV type 2 DNA with five restriction endonucleases.

The ordering of restriction endonuclease fragments of HSV-2 DNA for physical maps has been studied using molecular hybridization techniques and the cleavage of isolated restriction endonuclease fragments with further restriction endonucleases. Physical maps for the fragments produced by EcoRI, Hind III, Bgl II, Xba and Hpa I have been constructed. The mol. wt. of the various regions which constitute HSV-2 genome are very similar to the corresponding mol. wt. in the HSV-1 genome.