Robust Classical and Quantum Polarimetry with a Single Nanostructured Metagrating.

We formulate a new conceptual approach for one-shot complete polarization state measurement with nanostructured metasurfaces applicable to classical light and multiphoton quantum states by drawing on the principles of generalized quantum measurements based on positive operator-valued measures. Accurate polarization reconstruction from a combination of photon counts or correlations from several diffraction orders is robust with respect to even strong fabrication inaccuracies, requiring only a single classical calibration of the metasurface transmission. Furthermore, this approach operates with a single metagrating without interleaving, allowing for a reduction in metasurface size while preserving high transmission efficiency and output beam quality. We theoretically obtained original metasurface designs, fabricated the metasurface from amorphous silicon nanostructures deposited on glass, and experimentally confirmed accurate polarization reconstruction of laser beams. We also anticipate robust operation under changes in environmental conditions, opening new possibilities for space-based imaging and satellite optics.

The BAH domain, polybromo and the RSC chromatin remodelling complex.

The BAH (Bromo-adjacent homology) domain is a domain first identified in the vertebrate polybromo protein, a protein present in a large nuclear complex. Polybromo has two BAH domains, six bromodomains and an HMG-box. The BAH domain has been identified in a number of proteins involved in gene transcription and repression and is likely to be involved in protein-protein interactions. Polybromo resembles two related proteins in yeast, the Rsc1 and Rsc2 proteins, both having a BAH domain and two bromodomains as well as a DNA binding motif, the AT -hook. The Rsc1 and 2 proteins are components of the RSC (remodelling the structure of chromatin) complex and are required for transcriptional control. In this paper we review recent data on the function of the BAH and bromodomains in relation to polybromo and the Rsc proteins.

Molecular weight abnormalities of the CTCF transcription factor: CTCF migrates aberrantly in SDS-PAGE and the size of the expressed protein is affected by the UTRs and sequences within the coding region of the CTCF gene.

CTCF belongs to the Zn finger transcription factors family and binds to the promoter region of c-myc. CTCF is highly conserved between species, ubiquitous and localised in nuclei. The endogenous CTCF migrates as a 130 kDa (CTCF-130) protein on SDS-PAGE, however, the open reading frame (ORF) of the CTCF cDNA encodes only a 82 kDa protein (CTCF-82). In the present study we investigate this phenomenon and show with mass-spectra analysis that this occurs due to aberrant mobility of the CTCF protein. Another paradox is that our original cDNA, composed of the ORF and 3'-untranslated region (3'-UTR), produces a protein with the apparent molecular weight of 70 kDa (CTCF-70). This paradox has been found to be an effect of the UTRs and sequences within the coding region of the CTCF gene resulting in C-terminal truncation of CTCF-130. The potential attenuator has been identified and point-mutated. This restored the electrophoretic mobility of the CTCF protein to 130 kDa. CTCF-70, the aberrantly migrating CTCF N-terminus per se, is also detected in some cell types and therefore may have some biological implications. In particular, CTCF-70 interferes with CTCF-130 normal function, enhancing transactivation induced by CTCF-130 in COS6 cells. The mechanism of CTCF-70 action and other possible functions of CTCF-70 are discussed.

GATA-2 is a maternal transcription factor present in Xenopus oocytes as a nuclear complex which is maintained throughout early development.

We show that Xenopus oocytes and embryos contain GATA-2, stored in nuclei as a non-chromatin-bound complex. Its binding site specificity is different from that of GATA-1, having a much higher affinity for the motif with a core GATC sequence. This binding site preference was markedly reduced by either release of the factor with deoxycholate or purification on a DNA affinity column, suggesting a role for a cofactor(s). The identity of the maternal GATA factor was established as GATA-2 in two ways: (1) binding to an oligonucleotide probe was abolished by inclusion of either of two GATA-2 monoclonal antibodies, and (2) a protein of correct molecular weight for GATA-2 was detected by immunoblotting with a polyclonal antibody raised against a Xenopus GATA-2-specific peptide. Although predominantly complexed, some of the oocyte GATA-2 is functional as a transcription factor because the transcriptional activity of the chicken betaH-globin promoter injected into oocytes was reduced by mutation of either of two GATA binding sites. This effect was more pronounced when the stronger of the two sites was mutated. Butyrate treatment of oocytes stimulated cap-site initiation by up to 17-fold with both normal promoter and GATA site mutant constructs, showing that the mechanism of butyrate stimulation is not via GATA-2. The possible significance of regulating the availability of maternal GATA-2 during early development is discussed.

Molecular cloning of polybromo, a nuclear protein containing multiple domains including five bromodomains, a truncated HMG-box, and two repeats of a novel domain.

A number of transcription factors that act as adaptor proteins have been found to contain an 87 amino acid domain called the bromodomain. In a study to identify and characterise bromodomain proteins expressed in chicken cells, a novel gene has been isolated which encodes five repeats of the bromodomain. In addition, the encoded protein, termed polybromo, contains four other domains: an unusual truncated HMG box, two repeats of a novel domain which we term the BAH domain and a sequence related to a region within the regulatory domain of the DNA cytosine-5 methyltransferase enzyme. Polybromo was found to be related to a yeast protein U19102 which has two bromo domains, a BAH domain and the DNA methyltransferase-related sequence. Antibodies that were raised against polybromo were used in confocal microscopy analysis to show that the 180-kDa polybromo protein is located within the nucleus but excluded from the nucleolus. Gel filtration analysis of nuclear extracts demonstrate that polybromo is part of a large complex with a mass of approximately 2 million dalton.

CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms.

A novel sequence-specific DNA-binding protein, CTCF, which interacts with the chicken c-myc gene promoter, has been identified and partially characterized (V. V. Lobanenkov, R. H. Nicolas, V. V. Adler, H. Paterson, E. M. Klenova, A. V. Polotskaja, and G. H. Goodwin, Oncogene 5:1743-1753, 1990). In order to test directly whether binding of CTCF to one specific DNA region of the c-myc promoter is important for chicken c-myc transcription, we have determined which nucleotides within this GC-rich region are responsible for recognition of overlapping sites by CTCF and Sp1-like proteins. Using missing-contact analysis of all four nucleotides in both DNA strands and homogeneous CTCF protein purified by sequence-specific chromatography, we have identified three sets of nucleotides which contact either CTCF or two Sp1-like proteins binding within the same DNA region. Specific mutations of 3 of 15 purines required for CTCF binding were designed to eliminate binding of CTCF without altering the binding of other proteins. Electrophoretic mobility shift assay of nuclear extracts showed that the mutant DNA sequence did not bind CTCF but did bind two Sp1-like proteins. When introduced into a 3.3-kbp-long 5'-flanking noncoding c-myc sequence fused to a reporter CAT gene, the same mutation of the CTCF binding site resulted in 10- and 3-fold reductions, respectively, of transcription in two different (erythroid and myeloid) stably transfected chicken cell lines. Isolation and analysis of the CTCF cDNA encoding an 82-kDa form of CTCF protein shows that DNA-binding domain of CTCF is composed of 11 Zn fingers: 10 are of C2H2 class, and 1 is of C2HC class. CTCF was found to be abundant and conserved in cells of vertebrate species. We detected six major nuclear forms of CTCF protein differentially expressed in different chicken cell lines and tissues. We conclude that isoforms of 11-Zn-finger factor CTCF which are present in chicken hematopoietic HD3 and BM2 cells can act as a positive regulator of the chicken c-myc gene transcription. Possible functions of other CTCF forms are discussed.

Induction of differentiation of avian erythroblastosis virus-transformed erythroblasts by the protein kinase inhibitor H7: analysis of the transcription factor EF1.

The protein kinase inhibitor H7 [1-5(isoquinolinesulfonyl-2-methylpiperazine)] together with a temperature shift to 42 degrees C was found to reproducibly and efficiently induce differentiation of avian erythroblasts transformed with the avian erythroblastosis virus containing v-erbA and a temperature-sensitive v-erbB oncogene. Although a temperature shift to 42 degrees C without H7 results in some elevation of globin transcripts, much higher levels of transcripts accrue when cells are incubated at 42 degrees C with H7; under such conditions, 50-70% of the cells become benzidine positive. In order to investigate the mechanism by which the differentiation occurs, we have characterized and analyzed the levels of the erythroid transcription factor EF1, originally described as a factor binding to the beta H-globin promoter. Protein sequencing of EF1 shows that it is identical to the factor Eryf1. Using a peptide antibody and DNA-binding assays, we demonstrate that EF1 is present at high levels in the nucleus of undifferentiated HD3 cells, and, although there may be a small change when the cells are shifted to 42 degrees C, incubation of the cells with H7 at 42 degrees C does not result in a further elevation commensurate with the high levels of globin transcripts. It is concluded that v-erbA and v-erbB do not repress differentiation by limiting the levels of EF1.

A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene.

The chicken c-myc 5'-flanking sequence has previously been shown to bind multiple proteins present in undifferentiated and differentiated red blood cells. In this report the protein binding to one specific region within a hypersensitive site approximately 200 base pairs upstream of the start of transcription has been analysed in detail. Using a combination of a modified agarose gel retardation assay with O-phenanthroline-copper footprinting in situ, missing contact point and methylation interference techniques, two proteins were found to bind to overlapping sequences within 180-230 bp upstream of the start of transcription. One protein resembles the transcription factor Sp1, the other is a protein which binds to three regularly spaced repeats of the core sequence CCCTC. This CCCTC-binding factor was termed CTCF. It requires additional sequences outside the three recognition motifs for tight binding. CTCF was purified to near homogeneity by sequence-specific DNA chromatography. The approximate molecular weight of the CTCF was estimated to be 130,000. Removal of 110 bp sequence binding both CTCF and Sp1-like proteins leads to a 4 to 8-fold increase in transcription of stably transfected c-myc fusion constructs in chicken embryonic fibroblasts, suggesting that the CTCF is likely to be one of multiple nuclear factors involved in the transcriptional regulation of the chicken c-myc gene.

The purification of an erythroid protein which binds to enhancer and promoter elements of haemoglobin genes.

An erythroid nuclear protein (EF1), originally detected as a protein binding within the nuclease hypersensitive site upstream of the chicken beta H-globin gene, has been purified. This protein of 37,000-39,000 molecular weight binds to three sites within the hypersensitive region: one between the CCAAT and TATA boxes, the second (further upstream) next to a NF1 binding site, and the third adjacent to a regulatory element found in a number of beta-globin genes. The EF1 protein also binds to an erythroid-specific promoter element of the mouse alpha-globin gene and to two sites within the chicken beta A-globin enhancer. These six EF1-binding sites are related by the consensus sequence A/TGATAA/GG/C. A minor protein of molecular weight 72,000 which co-purifies with EF1 also binds to the same sequences.

TGGCA protein is present in erythroid nuclei and binds within the nuclease-hypersensitive sites 5' of the chicken beta H- and beta A-globin genes.

The developmentally regulated 5'-flanking DNase-I-hypersensitive site of the chicken beta H-globin gene in nuclei contains a subregion which is resistant to DNase I and which disappears when nuclei are extracted with 0.3 M NaCl, suggesting that there are salt-extractable proteins bound to sequences within this region. The 0.3 M NaCl extract contains two proteins which bind in vitro to these sequences. One of the binding sequences has an inverted repeat very similar to that bound by TGGCA protein. Partially purified TGGCA protein from chicken liver binds to this sequence in vitro giving exactly the same footprint as that obtained with erythroid nuclear proteins. Similarly TGGCA protein binds to an inverted repeat with the beta A-globin 5'-hypersensitive site giving a footprint identical to that obtained with erythroid nuclear protein extracts. From competition footprinting experiments and the electrophoretic mobility of the protein-DNA complex, it is concluded that the erythroid proteins previously described as binding to the beta H- and beta A-globin inverted repeats within the 5'-flanking hypersensitive sites both belong to the TGGCA protein family.

Elevated levels of a specific class of nuclear phosphoproteins in cells transformed with v-ras and v-mos oncogenes and by cotransfection with c-myc and polyoma middle T genes.

Transformation of a rat thyroid epithelial cell line (FRTL5-C12) with Kirsten and Harvey murine sarcoma viruses (carrying the ras oncogenes) results in elevated levels of three perchloric acid-soluble nuclear phosphoproteins. These three proteins are also induced to high levels in the PC-C13 thyroid epithelial cell line when transformed by the myeloproliferative sarcoma virus (carrying the v-mos oncogene) and when transformed by transfection with the c-myc proto-oncogene followed by infection with the polyoma leukaemia virus (PyMuLV) carry the polyoma middle T antigen gene. Neither c-myc or PyMuLV alone induced high levels of the three nuclear proteins. Untransformed thyroid fibroblasts have high levels of two of the three proteins and can be transformed by PyMuLV alone resulting in the appearance of the third protein. Transformation with Harvey sarcoma virus also results in the induction of the third protein. The three phosphoproteins have been purified by h.p.l.c. and shown to be related to the HeLa protein HMGI already described. The results of these studies indicate that elevated levels of these HMGI-like proteins are associated with neoplastic transformation and/or with an undifferentiated phenotype.

Characterisation of chicken erythroid nuclear proteins which bind to the nuclease hypersensitive regions upstream of the beta A- and beta H-globin genes.

Chicken erythrocyte sequence-specific nuclear DNA-binding proteins, which bind to the 5'-flanking DNAseI hypersensitive sites of the erythrocyte chromosomal beta A- and beta H-globin genes, have been fractionated by HPLC gel filtration. Three beta A-globin gene DNA binding activities (to sites A, B and B' (10-12)) were separated. The erythroid precursor cell line HD3 has beta A-globin gene sites B and B' binding activities, but binding to site A is detected only after the HD3 cells are induced to differentiate. The fractionated protein binds to a redefined site B', which contains at its center the globin CACCC consensus sequence. The chromosomal beta H-globin gene has two 5'-flanking DNAseI hypersensitive sites which bracket two sequences (H and H') bound by erythrocyte and HD3 nuclear protein in vitro. The beta H- and beta A-globin gene binding sites (H and B) contain variants of the sequences bound by Nuclear Factor 1 and the TGGCA-binding protein, and their protein binding activity(ies) co-purify after HPLC gel filtration.

Sequence-specific DNA-binding proteins which interact with (G + C)-rich sequences flanking the chicken c-myc gene.

The interaction of nuclear sequence-specific DNA-binding proteins from definitive chicken erythrocytes, thymus and proliferating transformed erythroid precursor (HD3) cells with the 700-base-pair (700-bp) DNA 5'-flanking region of the chicken c-myc gene was investigated by in vitro footprint analysis. The major HD3 protein-binding activity binds to a site (site V) 200 bp upstream from the 'cap' site but, after further fractionation, a second distinct binding activity is detected to a site (site VIII) which contains both the 'CAAT' and 'SP1-binding' consensus sequences. Protein from thymus and erythrocyte cells which express c-myc at lower levels, bind to seven and eight sites respectively. In common with HD3 cell protein, they both bind to site VIII and, although binding to the sequence at site V is also detected, the footprint protection pattern is sufficiently different (site V') to suggest the involvement of different proteins in terminally differentiated and proliferating cells. The DNA-binding activities were partially fractionated by high-performance liquid chromatography gel filtration and include an erythrocyte-specific protein which binds to a c-myc gene poly(dG) homopolymer sequence similar to that found upstream of the chicken beta A-globin gene.

Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strengths.

DNA sequence-specific binding proteins eluted from chicken erythrocyte and thymus nuclei, and fractionated as described by Emerson and Felsenfeld (19), have been investigated by filter binding and footprint analyses. The erythrocyte nuclear protein fraction specifically binds to at least two sites within the 5' flanking chromatin hypersensitive site of the chicken beta A-globin gene, and to a site 5' to the human beta-globin gene. The major chicken beta A globin gene binding site [G)18CGGGTGG) and the human beta-globin gene binding site [TA)6(T)8C(T)4) occur at or near sequences which are hypersensitive to S1 nuclease cleavage in supercoiled plasmids. Downstream, the second chicken beta A-globin gene binding site includes the beta-globin gene CACCC consensus sequence. Filter binding studies also show other sequence specific binding activities to human N-ras and human (but not chicken) c-myc gene sequences.

The effect of salt extraction on the structure of transcriptionally active genes; evidence for a DNAseI-sensitive structure which could be dependent on chromatin structure at levels higher than the 30 nm fibre.

The procedure developed by Lawson and Cole (Biochemistry, 1979, 18 2161-2166) for removing lysine-rich histones from nuclei at low pH also quantitatively extracts proteins HMG14 and 17. The effect of this low pH extraction on the DNAseI-sensitive structures of active genes in avian red blood cells has been investigated. No major perturbation of a developmentally regulated DNAseI hypersensitive site in the beta-globin domain and at the 5' end of the alpha D gene was seen. The overall DNAseI-sensitive conformation of the beta A-globin gene (relative to the ovalbumin gene) is minimally affected by pH3 salt extraction, but there is some loss of sensitivity of the alpha D gene. Removal of HMG proteins at neutral pH had no effect on the sensitivity of active genes in erythroid or fibroblast nuclei. These results, together with those carried out on DNAseI sensitivity and HMG binding to monomer nucleosomes, indicate that there is a major structural feature of active genes responsible for DNAseI-sensitivity which is independent of HMG proteins or nucleosome core particle structure but may be dependent on higher order chromatin structures.

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.

A quantitative analysis of histone H1 in rabbit thymus nuclei.

The relative quantity of histone H1 in rabbit thymus whole histone was determined to be 17.2% (w/w). This implies that there is, on average, one histone H1 molecule per nucleosome.

An improved large scale fractionation of high mobility group non-histone chromatin proteins.

1. Methodology is presented for the large scale preparation and fractionation of high mobility group proteins from calf thymus chromatin. The total high mobility group protein from approx. 1 kg calf thymus tissue can be separated into five fractions by CM-Sephadex C25 ion-exchange chromatography. High mobility group proteins 1 and 2 comprise two fo the fractions. From a third fraction two more chromatin proteins, protein 3 and 17, can be isolated by trichloroacetic acid precipitation and CM-cellulose chromatography at pH 5.5. 2. The four proteins thus purified are lysine-rich proteins. Proteins 1 and 2 are additionally characterised by their high contents of acidic amino acids, as described previously (Goodwin, G. H. and Johns, E. W. (1973) Eur. J. Biochem. 40, 215-219). Proteins 3 and 17, having lower contents of acidic amino acids, are basic proteins similar to the histones. All four proteins exhibit single N-terminal amino acids; glycine is the N-terminal group of proteins 1, 2 and 3; protein 17 has a proline N-terminal amino acid. The proteins are not highly phosphorylated nor are they associated with appreciable quantities of nucleic acid.