Organelle positioning in muscles requires cooperation between two KASH proteins and microtubules

Striated muscle fibers are characterized by their tightly organized cytoplasm. Here, we show that the Drosophila melanogaster KASH proteins Klarsicht (Klar) and MSP-300 cooperate in promoting even myonuclear spacing by mediating a tight link between a newly discovered MSP-300 nuclear ring and a polarized network of astral microtubules (aMTs). In either klar or msp-300(ΔKASH), or in klar and msp-300 double heterozygous mutants, the MSP-300 nuclear ring and the aMTs retracted from the nuclear envelope, abrogating this even nuclear spacing. Anchoring of the myonuclei to the core acto-myosin fibrillar compartment was mediated exclusively by MSP-300. This protein was also essential for promoting even distribution of the mitochondria and ER within the muscle fiber. Larval locomotion is impaired in both msp-300 and klar mutants, and the klar mutants were rescued by muscle-specific expression of Klar. Thus, our results describe a novel mechanism of nuclear spacing in striated muscles controlled by the cooperative activity of MSP-300, Klar, and astral MTs, and demonstrate its physiological significance.     

Synergism between two distinct elements of the HTLV-I enhancer during activation by the trans-activator of HTLV-I

We have conducted functional studies of the enhancer elements of human T-cell leukemia virus type I (HTLV-I) using the human T-cell lines Jurkat and MOLT 4, which are negative for HTLV-I, and MT-2 and TL-Mor, which carry the proviral genome of HTLV-I. Two distinct elements have been implicated in function of the HTLV-I enhancer. One is the 21-base-pair (bp) core element that is responsible for trans-activation by the HTLV-I trans-activator p40tax and that has the ability to bind to cyclic-AMP responsive element binding factor (CREB)-like factor(s). The other is a region interposed between the 21-bp elements. In this study we demonstrate that a subfragment (C26) in the region between the 21-bp elements is involved in trans-activation by p40tax, possibly through binding to an NF-kappa B-like nuclear factor or factors. Formation of the protein-DNA complex with the C26 subfragment was positively affected by p40tax. The C26 element conferred partial responsiveness to p40tax when linked to one copy of the 21-bp element that, by itself, showed little activation in response to p40tax. However, the C26 element alone, even when repeated, could not be activated by p40tax, unlike other NF-kappa B-binding elements. In contrast, the C26 element itself was profoundly activated upon stimulation with 12-O-tetradecanoylphorbol-13-acetate. These findings therefore suggest that the HTLV-I enhancer contains multiple functional elements, including binding sites for at least CREB- and NF-kappa B-like factors, which synergistically cooperate in activation of the HTLV-I enhancer in response to p40tax. Our results also demonstrate that TPA-dependent activation of the HTLV-I enhancer may be mediated through the C26 element.     

Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.

The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.     

Two cellular single-strand-specific DNA-binding proteins interact with two regions of the bovine papillomavirus type 1 genome, including the origin of DNA replication.

We have identified and purified to near homogeneity two specific single-stranded DNA-binding factors (SPSF I and II) with molecular masses of 42 and 39 kDa, respectively, from calf thymus. Gel retention analysis and competition experiments demonstrate that the ubiquitous proteins SPSF I and II specifically interact with single-stranded DNA derived from the minimal in vitro origin of replication of bovine papillomavirus type 1 and a region of the viral genome proposed to be involved in plasmid maintenance. Bovine papillomavirus type 1 proteins do not interfere with DNA binding of SPSF I and II. The exact location of the binding domains of SPSF I and II on the DNA has been determined by methylation interference and T4 DNA polymerase footprinting. A potential cellular binding site for SPSF I and II is the major promoter (P2) of the human c-myc gene.     

Functional analysis of the murine T-cell receptor beta enhancer and characteristics of its DNA-binding proteins.

The minimal T-cell receptor (TCR) beta-chain (TCR beta) enhancer has been identified by transfection into lymphoid cells. The minimal enhancer was active in T cells and in some B-lineage cells. When a larger fragment containing the minimal enhancer was used, its activity was apparent only in T cells. Studies with phytohemagglutinin and 4 beta-phorbol-12,13-dibutyrate revealed that the enhancer activity was increased by these agents. By a combination of DNase I footprinting, gel mobility shift assay, and methylation interference analysis, seven different motifs were identified within the minimal enhancer. Furthermore, competition experiments showed that some of these elements bound identical or similar factors that are known to bind to the TCR V beta promoter decamer or to the immunoglobulin enhancer kappa E2 or muEBP-E motif. These shared motifs may be important in the differential gene activity among the different lymphoid subsets.     

Binding specificity of the two major DNA-binding proteins in human serum.

The two major DNA-binding proteins of human serum (DNA-binding protein 1 and DNA-binding protein 2) were shown to bind preferentially to single-stranded polynucleotides rich in guanine residues. Equilibrium competition experiments using a nitrocellulose filter assay system containing labeled human lymphocyte DNA and various competing natural and synthetic polynucleotides indicated that both proteins recognized sequences of bases containing a keto group in either position 6 (purines) or 4 (pyrimidines) and that these keto groups must be readily accessible for effective binding to occur. Guanine was shown to be the preferred nucleotide through inhibition experiments using a series of synthetic homopolymers and a series of bacterial DNAs of differing G + C content. The relationship between protein affinity and G + C content was shown to be directly proportional. The equilibrium constants for the binding of the human lymphocyte DNA by both proteins were on the order of 10(-6) M, and the length of the nucleotide sequence necessary for effective binding was found to be 12 to 18 bases using a series of oligomers of poly(dG).     

Synthesis of linear gramicidin requires the cooperation of two independent reductases

The linear pentadecapeptide gramicidin has been reported to be assembled by four large multimodular nonribosomal peptide synthetases (NRPSs), LgrABCD, that comprise 16 modules. During biosynthesis, the N-formylated 16mer peptide is bound to the peptidyl carrier protein (PCP) of the terminal module via a thioester bond to the carboxyl group of the last amino acid glycine(16). In a first reaction the peptide is released from the protein template in an NAD(P)H-dependent reduction step catalyzed by the adjacent reductase forming an aldehyde intermediate. Here we present the biochemical proof that this aldehyde intermediate is further reduced by an aldoreductase, LgrE, in an NADPH-dependent manner to form the final product gramicidin A, N-formyl-pentadecapeptide-ethanolamine. To determine the potential use of the two reductases in the construction of hybrid NRPSs, we have tested their ability to accept a variety of different substrates in vitro. The results obtained give way to a broad spectrum of possible use.     

Soluble HTLV-I Tax1 protein stimulates proliferation of human peripheral blood lymphocytes.

Human T-cell leukemia virus type I (HTLV-I) is associated with two human diseases, adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). Lymphocytes from patients with ATL or TSP/HAM display abnormal proliferation properties in culture. Here we report that purified, soluble Tax1 protein can be taken up by, and stimulate proliferation of, uninfected human peripheral blood lymphocytes (PBLs) that have been stimulated with phytohemagglutinin (PHA). Tax1 was 40 to 70% as active as interleukin-2 (IL-2) in stimulating proliferation of PBLs. Heat inactivation, chloroform extraction, and immunoprecipitation with antisera specific for Tax1 each abolished the ability of the protein to stimulate lymphocyte proliferation. Tax1 failed to stimulate PBL proliferation in the absence of PHA. After an initial round of cell division, Tax1-treated PBLs exhibited prolonged sensitivity to IL-2-induced proliferation. These results indicate that Tax1 can stimulate lymphocyte proliferation in culture and imply that extracellular Tax1 may be involved in the spontaneous proliferation of TSP/HAM lymphocytes and the IL-2-dependent proliferation of ATL lymphocytes.     

Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA.

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.