A structure of potentially active and inactive genes of chicken erythrocyte chromatin upon decondensation.

In the presence of 3 mM MgCl2 DNase I cleavage of bulk, globin and ovalbumin gene chromatin in chicken erythrocyte nuclei generates fragments which are multiples of a double-nucleosome repeat. However, in addition to the dinucleosomal periodicity beta-globin gene chromatin was fragmented into multiples of a 100 b.p. interval which is characteristic for partially unfolded chromatin. This distinction correlates with higher sensitivity of beta-globin domain to DNase I and DNase II as compared to the inactive ovalbumin gene. At 0.7 mM MgCl2 where these DNases fragment bulk chromatin into series of fragments with a 100 b.p. interval, the difference in digestibility of the investigated genes is dramatically decreased. When chromatin has been decondensed by incubation of nuclei in 10 mM Tris-buffer, DNase II generates a typical nucleosomal repeat, and the differential nuclease sensitivity of the analyzed genes is not observed. The data suggest that higher nuclease sensitivity of potentially active genes is due to irregularities in higher order chromatin structure.     

Affinity chromatography of DNA labeled with chemically cleavable biotinylated nucleotide analogs

DNA labeled with the chemically cleavable biotinylated nucleotide Bio-12-SS-dUTP was chromatographed on biotin cellulose affinity columns using either avidin or streptavidin as the affinity reagent. Although both proteins were equally effective in binding the Bio-12-SS-DNA to the affinity resin, two important differences were found. First, nonbiotinylated DNA bound to avidin, but not to streptavidin, in buffers containing 50 mM NaCl. Second, Bio-12-SS-DNA was released much more slowly from the streptavidin affinity column than from the avidin column upon washing with buffer containing dithiothreitol. This difficulty in reducing the disulfide bond of Bio-12-SS-DNA bound to streptavidin is most likely due to steric protection of the disulfide bond by the protein. This conclusion is supported by our finding that DNA labeled with another biotinylated nucleotide analog, Bio-19-SS-dUTP, is rapidly and efficiently recovered from a streptavidin column. In Bio-19-SS-DNA, the distance between the disulfide bond and the biotin group is approximately 10 A greater than that in Bio-12-SS-DNA. Therefore, Bio-19-SS-dUTP and streptavidin form the basis of an efficient affinity system for the isolation and subsequent recovery of biotinylated DNA in the presence of low ionic strength buffers.     

Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II

We have examined in some detail the chromatin structure of a 6.2 kilobase pair (kbp) chromosomal region containing the chicken beta-globin gene. The chromatin structure was probed with three nucleases, DNase I, micrococcal nuclease, and DNase II, and the rate of digestion of specific subfragments of the region was compared with the rate of bulk DNA digestion. We have characterized the rate of digestion of each fragment in terms of a sensitivity factor which measures the sensitivity of a fragment to a particular nuclease relative to bulk DNA. The sensitivity factors were determined by a least squares curve fitting method based on target analysis. In nuclei isolated from 14-day-old chicken embryo red blood cells, the entire 6.2-kbp region shows approximately a 10- to 20-fold increase in sensitivity to DNase I, a 3-fold increased sensitivity to micrococcal nuclease, and a 6-fold increased sensitivity to DNase II. In addition to the adult beta-globin gene, this region contains 5' and 3' flanking sequences, the 5' half of the inactive, embryonic globin gene, epsilon, and some repeated sequences. There is no obvious correlation between these genetic elements and the overall chromatin structure as measured by the nuclease sensitivity. This same region shows little or no special sensitivity in nuclei isolated from 14-day-old chicken embryo brain. Furthermore, fragments of the inactive ovalbumin gene show little or no sensitivity in either red blood cells or brain. These results support the conclusion that the entire 6.2-kbp region is largely packaged as active chromatin in 14-day-old chicken embryo red blood cells.     

Activated murine alpha-globin gene is not preferentially associated with the nuclear matrix

The association of the murine alpha-globin gene with the nuclear matrix was studied in three different states of the gene: inactive (EAT cells), potentially active (MEL cells) and active (induced MEL cells). When "native" nuclei were digested with DNase I it was found that the nuclear matrix was not enriched in alpha-globin DNA sequences in all three different types of cells. A nuclease-hypersensitive site in the 5'-flanking region of the alpha-globin gene was detected in the induced MEL-cells.     

Preferential in vitro binding of high mobility group proteins 14 and 17 to nucleosomes containing active and DNase I sensitive single-copy genes

High mobility group (HMG) proteins 14 and 17 bind to mononucleosomes in vitro, but the exact nature of this binding has not been clearly established. A new method was developed to allow direct membrane transfer of DNA from HMG 14/17 bound and unbound nucleosomes, which have been separated by acrylamide gel electrophoresis. Hybridization analysis of membranes obtained by this method revealed that the HMG 14/17 bound nucleosomes of avian erythrocytes and rat hepatic tumor (HTC) cells were enriched, about 2-fold, in actively transcribed genes and also inactive but DNase I sensitive genes. Nucleosomes containing inactive, DNase I resistant genes were bound by HMG 14/17, but not preferentially. Several factors that have been reported to greatly influence the binding of HMG 14/17 to nucleosomes in vitro were tested and shown to not account for the preferential binding to DNase I sensitive chromatin. These factors include nucleosomal linker DNA length, single-stranded DNA nicks, and DNA bulk hypomethylation. An additional factor, histone acetylation, was preferentially associated with the HMG 14/17 bound chromatin fraction of avian erythrocytes, but it was not associated with the HMG 14/17 bound chromatin fraction of metabolically active HTC cells. The latter finding was true for all kinetic forms of histone acetylation.     

Structural state of active and inactive genes during chromatin decondensation

Chromatin structure of globin and ovalbumin genes in chicken erythrocyte nuclei has been investigated by means of the "nuclease criterion" (described earlier). In intact nuclei (i.e. in the presence of 3 mM MgCl2) DNase I cleaves chromatin of both genes generating fragments multiple of a double-nucleosome repeat (2N-periodicity). However, in the case of the globin gene, apart from the 2N-periodicity, fragments were observed that are multiple of 100 b.p. and are characteristic for partially unfolded chromatin. This distinction in nuclease cleavage patterns correlates with a higher sensitivity of the globin gene as compared with the inactive ovalbumin gene. At 0.5-0.7 mM MgCl2 the transition from dinucleosomal fragmentation with DNase I and DNase II to fragmentation via a 100 b.p. interval occurs and the difference in digestibility of both genes is dramatically increased. If chromatin has been decondensed by incubation of nuclei in 10 mM Tris-buffer DNase Il generates an usual nucleosomal repeat, and in this ionic conditions one may not observe any difference in nuclease sensitivity of the analyzed genes. The data allow to suggest that the high nuclease sensitivity of potentially active genes can be conditioned by more relaxed arrangement of nucleosomes in higher order chromatin structure.     

Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4) functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4) keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8). This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8) is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off) of 4.28×10(-4) s(-1) and K(d) of 1.9×10(-8) M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.     

The nuclease sensitivity of active genes.

Brief micrococcal nuclease digestion of chick embryonic red blood cells results in preferential excision and solubilization of monomer nucleosomes associated with beta-globin sequences and also 5'-sequences flanking the beta-globin gene. Both regions are DNAse-I sensitive in nuclei. Such salt-soluble nucleosomes are enriched in all four major HMG proteins but HMG1 and 2 are only weakly associated. These nucleosomes appear to have lost much of the DNAse-I sensitivity of active genes. The HMG14 and 17-containing salt-soluble nucleosomes separated by electrophoresis are not DNAse-I sensitive and contain inactive gene sequences as well as active sequences. Reconstitution of HMG proteins onto bulk nucleosomes or chromatin failed to reveal an HMG-dependent sensitivity of active genes as assayed by dot-blot hybridization and it was found that the DNAse-I sensitivity of ASV proviral sequences as assayed by dot-blot hybridization was not HMG-dependent. These results indicate that higher order chromatin structures might be responsible for nuclease sensitivity of active genes.     

The content of various active and inactive genes in chromatin fractions with different accessibilities of DNA to polycyclic aromatic hydrocarbons

Nuclear chromatin was separated into four fractions according to solubility in low (0.2 mM MgCl2, 10 mM Tris-HCl, pH 7.6) and high (2 M NaCl) ionic strengths after digestion of nuclear DNA with nucleases. In nuclear matrix DNA the ratio of active to inactive genes was always higher than that in the original total DNA, i.e., 25 times greater in rat liver nuclei. In DNA released from the nuclei into a low ionic strength buffer the active to inactive gene ratio was lower than in total DNA (3.7 times as low in case of rat liver nuclei). The amount of carcinogens in matrix DNA exceeded that of DNA soluble in a low ionic strength buffer (3-4 times in case of rat liver nuclei and 16 times in case of hamster embryo cells). The two other fractions occupied an intermediate position between the above said fractions of DNA. The experimental results suggest that the level of carcinogen-induced modifications may be increased in active genes, including transcribed protooncogenes.