Chromatin structure of altered yeast centromeres.

We have investigated the chromatin structure of wild-type and mutationally altered centromere sequences in the yeast Saccharomyces cerevisiae by using an indirect end-labeling mapping strategy. Wild-type centromere DNA from chromosome III (CEN3) exhibits a nuclease-resistant chromatin structure 220-250 base pairs long, centered around the conserved centromere DNA element (CDE) III. A point mutation in CDE III that changes a central cytidine to a thymidine and completely disrupts centromere function has lost the chromatin conformation typically associated with the wild-type centromere. A second conserved DNA element, CDE I, is spatially separated from CDE III by 78-86 A + T-rich base pairs, which is termed CDE II. The sequence and spatial requirements for CDE II are less stringent; alterations in CDE II length and sequence can be tolerated to a limited extent. Nuclease-resistant cores are altered in dimension in two CDE II CEN3 mutations. Two CDE I deletion mutations that retain partial centromere function also show nuclease-resistant regions of reduced size and intensity. The results from a number of such altered centromeres indicate a correlation between the presence of a protected core and centromere function.     

Murine Hox-1.11 homeobox gene structure and expression.

The Hox-1.11 gene encodes a protein 372 amino acid residues long that contains a conserved pentapeptide, a homeodomain, and an acidic region. The amino acid sequence of the homeodomain of Hox-1.11 is identical to that of Hox-2.8, and the N-terminal and C-terminal regions of Hox-1.11 are similar to those of human HOX2H, which is the equivalent of murine Hox-2.8. The Hox-1.11 gene was shown to reside on murine chromosome 6, which contains the Hox-1 cluster of homeobox genes. One species of Hox-1.11 poly(A)+ RNA approximately 1.7 kb long was detected in mouse embryos, which is most abundant in 12-day-old embryos and progressively decreases during further embryonic development. The most anterior expression of Hox-1.11 poly(A)+ RNA in 12- to 14-day-old mouse embryos was shown by in situ hybridization to be in the mid and posterior hindbrain. Hox-1.11 poly(A)+ RNA also is expressed in the VII and VIII cranial ganglia, spinal cord, spinal ganglia, larynx, lungs, vertebrae, sternum, and intestine.     

Chromatin structure in the cellular slime mold Dictyostelium discoideum.

The structure of Dictyostelium discoideum chromatin has been studied by the following techniques: electron microscopy, staphylococcal nuclease digestion, acrylamide gel electrophoresis, sucrose gradient centrifugation, and melting. The basic unit of chromatin is the nucleosome, which is a particle 98.6 A in diameter. Approximately 50% of the chromatin is protected from nuclease digestion, but this decreases when protease activity is not inhibited. The nucleosome contains 187 base pairs of DNA, including a 137-base-pair core and a 50-base-pair linker. The monomer nucleosome has an s20,w value of 11.5 S on isokinetic sucrose gradients. When the chromatin is melted, four transitions are observed, at 54.5 degrees, 66.7 degress, 74.9 degrees, and 79.7 degrees. The structure of Dictyostelium chromatin is very similar to that seen in higher eukaryotes.     

Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum.

The enzyme dihydropteroate synthetase (DHPS) from Plasmodium falciparum is involved in the mechanism of action of the sulfone/sulfonamide group of drugs. We describe the cloning and sequencing of the gene encoding the P. falciparum DHPS enzyme and show that it is a bifunctional enzyme that includes dihydro-6-hydroxymethylpterin pyrophosphokinase (PPPK) at the N terminus of DHPS. The gene encodes a putative protein of 83 kDa that contains two domains that are homologous with the DHPS and PPPK enzymes of other organisms. The PPPK-DHPS gene is encoded on chromosome 8 and has two introns. An antibody raised to the PPPK region of the protein was found to recognize a 68-kDa protein that is expressed throughout the asexual life cycle of the parasite. We have determined the sequence of the DHPS portion of the gene from sulfadoxine-sensitive and -resistant P. falciparum clones and identified sequence differences that may have a role in sulfone/sulfonamide resistance.     

Chromatin higher-order structure studied by neutron scattering and scanning transmission electron microscopy.

Neutron scattering in solution and scanning transmission electron microscopy were simultaneously done on chicken erythrocyte chromatin at various salt and magnesium concentrations. We show that chromatin is organized into a higher-order structure even at low ionic strength and that the mass per unit length increases continuously as a function of salt concentration, reaching a limiting value of between six and seven nucleosomes per 11 nm. There is no evidence of a transition from a 10-nm to a 30-nm fiber. Fiber diameter is correlated with mass per unit length, showing that both increase during condensation. We also find that there is no essential difference between the mass per unit length measured by scanning transmission electron microscopy and neutron scattering in solution, showing that the ordered regions seen in micrographs are representative of chromatin in solution.     

气道黏蛋白的结构及其基因表达的调节

黏蛋白（mucin,MUC）是具有特征性串联重复区（tandem repeat,TR）区域的糖蛋白。这些TR区域富含丝氮酸或苏氯酸以及脯氨酸。MUC骨架由MUC基因编码．其中已有20种人类MUC基因存最近二十年通过DNA重组技术被确认。大多数TR丝氨酸／苏氨酸残基都被O-糖基化。MUC5AC和MUC5B是气道中最主要的两种MUC,它们的TR在蛋白质骨架中分别重复4、5倍。这些重复的TR被富集的半胱氯酸分隔．其中每个TR包含脯氮酸、丰富的丝／苏氨酸残基和O-糖基化位点,TR序列可重复成百上千．而每个TR中可含有数百个O-糖链结合位点．故MUC蛋白质骨架的TR数目决定MUC大小,并且O-糖链的添加可使MUC构型发生改变。因此TR可直接或间接地造成MUC构象和功能的多样性。TR和连接的O-糖链可影响MUC的大小、形状和质量．从而形成黏液本身不同的生物物理学和生物学性质。MUC是黏膜免疫学的先天性防御系统,这个观点．在不断地得到更多的支持。MUCSAC和MUCSB足健康气道黏液和慢性气道疾病患苦痰液的主要组分。几种炎症性气道疾病有共同的途径：MUC基因表达增加、杯状细胞增生和（或）黏膜下腺肥大。每个过程都可能造成支气管哮喘、慢性阻塞性肺疾病或囊性纤维病患者气道MUC过度产生和黏液梗阻。过去十年里,我们对这些变化的病理生理机制研究已有了显著进步。相关因子如生长因子、免疫／炎症介质、环境刺激因素、病原等可通过丝裂原激活的蛋白激酶、表皮中长因子受体等不同的细胞信号转导通路在转录（mRNA）和（或）转录后水平（如精基化）对其进行调节有差别地调节MUC基因表达。     

Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization.

Thrombopoietin (TPO), a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells, is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe, we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure similar to that of the erythropoietin gene (EPO). Southern blot analysis of human genomic DNA reveals a hybridization pattern consistent with a single gene locus. The locus was mapped by in situ hybridization of metaphase chromosome preparations to chromosome 3q26-27, a site where a number of chromosomal abnormalities associated with thrombocythemia in cases of acute myeloid leukemia have been mapped. A human TPO cDNA was isolated by PCR from kidney mRNA. The cDNA encodes a protein with 80% identity to previously described murine TPO and is capable of initiating a proliferative signal to murine interleukin 3-dependent BaF3 cells expressing the murine or human TPO receptor.     

Chromatin structure analysis of the mouse Xist locus

The Xist gene is expressed exclusively from the inactive X chromosome and plays a central role in regulating X chromosome inactivation. Here we describe experiments aimed at defining the extent of the active chromatin domain of the expressed Xist allele. By using an allele-specific general DNaseI sensitivity assay we show that there is preferential digestion of the expressed allele at sites within the transcribed locus but not in flanking sites located up to 70 kb 5'. A putative proximal boundary for the Xist domain is located within 10 kb upstream of promoter P1. Chromatin in the expressed domain was found to be acetylated at H4 in XX somatic cells but also in XY cells, where Xist is never expressed. A single clear exception to this was the Xist promoter, which is acetylated only in XX cells. These observations concur with the view that H4 acetylation may not be a general marker of active chromatin domains and further support data implicating local promoter acetylation as being of primary functional significance in vivo.     

An erythroid-specific chromatin opening element reorganizes beta-globin promoter chromatin structure and augments gene expression.

In erythroid tissues the chromatin structure of the beta-globin gene locus is extensively remodeled. Changes include the formation of DNase I hypersensitive sites (HSs) over the promoters of actively expressed genes. To test the hypothesis that such "opening" of promoter chromatin structure is important for beta-globin gene expression, we placed a 101-bp erythroid-specific hypersensitive-site forming element (HSFE) from the core of LCR HS4 immediately upstream of a minimal beta-globin gene promoter. We then studied the effects of this element alone and in combination with other cis-acting elements on globin gene chromatin structure and gene expression in MEL cells and transgenic mice. Single or tandem HSFEs increased the size of the portion of the promoter accessible to DNase digestion, increased the proportion of promoters in an accessible conformation, and increased gene expression approximately 5-fold. These were equivalent to expression levels attained using a 2.8-kb microLCR construct. Inclusion of the LCR HS2 enhancer did not increase expression further. In transgenic mouse fetal liver cells the HSFE increased average expression 2.5-fold compared to the minimal promoter alone. These results indicate that a small cis-acting element is capable of remodeling local beta-globin promoter chromatin structure and producing expression similar to that seen with a microLCR construct.