Independently regulated neocentromere activity of two classes of tandem repeat arrays

Tandem repeat arrays often are found in interstitial (i.e., normally gene-rich) regions on chromosomes. In maize, genes on abnormal chromosome 10 induce the tandem repeats that make up knobs to move poleward on the meiotic spindle. This so-called neocentromere activity results in the preferential recovery, or meiotic drive, of the knobs in progeny. Here we show that two classes of repeats differ in their capacity to form neocentromeres and that their motility is controlled in trans by at least two repeat-specific activators. Microtubule dynamics appear to contribute little to the movement of neocentromeres (they are active in the presence of taxol), suggesting that the mechanism of motility involves microtubule-based motors. These data suggest that maize knob repeats and their binding proteins have coevolved to ensure their preferential recovery in progeny. Neocentromere-mediated drive provides a plausible mechanism for the evolution and maintenance of repeat arrays that occur in interstitial positions.     

Inhibition of rat growth hormone promoter activity by site-specific DNA methylation

The effect of methylation on rat growth hormone (rGH) promoter activity was determined in GH3 cells by measuring rGH-Neo and rGH-CAT fusion gene expression with or without prior in vitro treatment with the site-specific DNA methyltransferases, M-BsuE and M-HhaI. To assay for rGH-promoter-specific effects of DNA methylation, RSV-Neo and RSV-CAT activities with or without M-BsuE, M-HhaI and M-HpaII treatment were measured in parallel cultures of GH3 cells. GH1-Neo and RSV-Neo fusion gene expression was inhibited by in vitro methylation from 44 to 83% as measured by the number of Geneticin-resistant GH3 cell colonies. Methylation of the GH1 promoter by M-BsuE exhibited some selective inhibition of Neo expression as determined by colony numbers, although extensive methylation of non-promoter DNA in GH1-Neo and RSV-Neo by M-HhaI and M-HpaII also inhibited Neo expression. Southern blot analysis of genomic DNA isolated from the Geneticin-resistant GH3 cells indicated that Geneticin-resistance was accompanied by demethylation of the BsuE (ThaI) sites in stably incorporated GH1-Neo DNA but not RSV-Neo DNA. Transient expression of the CAT gene in GH3 cells was selectively inhibited by 60% upon methylation of two BsuE (ThaI) sites in the GH1 promoter of GH1-CAT by M-BsuE. The data demonstrate, for the first time, to our knowledge, a direct effect of DNA methylation on the activity of the rat growth hormone promoter.     

DNA photorepair: chromophore composition and function in two classes of DNA photolyases

DNA photolyase catalyzes the repair of pyrimidine dimers in UV-damaged DNA in a reaction which requires visible light. Class I photolyases (Escherichia coli, yeast) contain 1,5-dihydroFAD (FADH2) plus a pterin derivative (5,10-methenyltetrahydropteroylpolyglutamate). In class II photolyases (Streptomyces griseus, Scenedesmus acutus, Anacystis nidulans, Methanobacterium thermoautotrophicum) the pterin chromophore is replaced by an 8-hydroxy-5-deazaflavin derivative. The two classes of enzymes exhibit a high degree of amino acid sequence homology, suggesting similarities in protein structure. Action spectra studies show that both chromophores in each enzyme tested act as sensitizers in catalysis. Studies with E. coli photolyase show that the pterin chromophore is not required when FADH2 acts as the sensitizer but that FADH2 is required when the pterin chromophore acts as sensitizer. FADH2 is probably the chromophore that directly interacts with substrate in a reaction which may be initiated by electron transfer from the excited singlet state (1FADH2*) to form a flavin radical plus an unstable pyrimidine dimer radical. Pterin, the major chromophore in E. coli photolyase, may act as an antenna to harvest light energy which is then transferred to FADH2.     

A short repeat in the genome of the DNA sequences flanking bovine growth hormone genes

The phage clones containing a gene coding for bovine growth hormone were isolated from a bovine genomic library. Comparison of the 5' and 3' regions flanking the bovine growth hormone gene by Southern blot hybridization revealed that they share homology. Screening the bovine genomic library by nick-translated DNA fragment from 5' flanking region leads to conclusion that this sequence is present in 0.1% of clones. Each analysed clone carrying the sequence contains some copies of it.     

Human growth hormone DNA sequence and mRNA structure: possible alternative splicing.

We have determined the complete sequence of the human growth hormone (hGH) gene and the position of the mature 5' end of the hGH mRNA within the sequence. Comparison of this sequence with that of a cloned hGH cDNA shows that the gene is interrupted by four intervening sequences. S1 mapping shows that one of these intervening sequences has two different 3' splice sites. These alternate splicing pathways generate hGH peptides of different sizes which are found in normal pituitaries. Comparison of sequences near the 5' end of the hGH mRNA with a similar region of the alpha subunit of the human glycoprotein hormones reveals an unexpected region of homology between these otherwise unrelated peptide hormones.     

Interspersion of repetitive sequences in rat liver DNA

In rat liver DNA, which contains only 20% repetitive sequences, a close interspersion of repetitive and unique sequences is found in about 35 % of the total DNA. The mean length of repetitive and unique alternating sequences is respectively 230 and 400 base pairs.     

Thyroid hormone and growth: relationships with growth hormone effects and regulation

For some years, research in the field of growth endocrinology has been mainly focused on growth hormone (GH). However, it appears that GH does not always control growth rate. For instance, it does not clearly influence intra-uterine growth: moreover, although the results of GRF or GH administration appear convincing in rats, pigs or heifers, this is not the case in chickens and lambs. In addition, GH does not always clearly stimulate somatomedin production, particularly diring food restriction and fetal life, and in hypothyroid animals or sex-linked dwarf chickens. In such situations, this phenomenon is associated with a reduced T3 production, suggesting a significant influence of thyroid function on GH action, and more generally, on body growth. In fact, numerous data demonstrate that thyroid hormone is strongly involved in the regulation of body growth. In species with low maturity at birth, such as the rat. T4 and T3 affect postnatal growth eleven days earlier than the appearance of GH influence. In contrast to GH, thyroid hormone significantly influences fetal growth in sheep. Moreover, the body growth rate is clearly stimulated by T3 in dwarf animals. In addition to its complex metabolic effects involved in the general mechanisms of body growth, thyroid hormone stimulates the production of growth factors, particularly EGF and NGF. Moreover, it affects GH and somatomedin production and also their tissue activity. All these results strongly suggest that it would be difficult to study GH regulation and physiological effects without taking thyroid function into account.     

Structures of two classes of MHC molecules elucidated: crucial differences and similarities

New structures of MHC molecules have significantly improved our understanding of molecular recognition in cellular immunology. Highlights include the first structure of a class II MHC molecule, complexed with a viral peptide and with a bacterial superantigen. A structure of an MHC-like Fc receptor is expected soon. Interesting comparisons can now be made between the recognition properties of MHC and MHC-like proteins.     

Autocrine growth hormone: effects on growth hormone receptor trafficking and signaling

GH and GH receptor are expressed in many extrapituitary tissues, permitting autocrine/paracrine activity. Autocrine GH has regulatory functions in embryonic development and cellular differentiation and proliferation and is reported to be involved in the development and metastasis of tumor cells. To understand the principles of transport and signaling of autocrine GH and GH receptor, we used a model system to express both proteins in the same cell. Our experiments show that GH binds the GH receptor immediately after synthesis in the endoplasmic reticulum and facilitates maturation of GH receptor. The hormone-receptor complexes arrive at the cell surface where exogenously added GH is unable to bind these receptors. Autocrine GH activates the GH receptors, but signal transduction occurs only after exiting the endoplasmic reticulum. This model study explains why autocrine GH-producing cells may be insensitive for GH (antagonist) treatment and clarifies autocrine signaling events.     

Epigenetics of a tandem DNA repeat: chromatin DNaseI sensitivity and opposite methylation changes in cancers

DNA methylation and chromatin DNaseI sensitivity were analyzed in and adjacent to D4Z4 repeat arrays, which consist of 1 to ~100 tandem 3.3-kb units at subtelomeric 4q and 10q. D4Z4 displayed hypomethylation in some cancers and hypermethylation in others relative to normal tissues. Surprisingly, in cancers with extensive D4Z4 methylation there was a barrier to hypermethylation spreading to the beginning of this disease-associated array (facioscapulohumeral muscular dystrophy, FSHD) despite sequence conservation in repeat units throughout the array. We infer a different chromatin structure at the proximal end of the array than at interior repeats, consistent with results from chromatin DNaseI sensitivity assays indicating a boundary element near the beginning of the array. The relative chromatin DNaseI sensitivity in FSHD and control myoblasts and lymphoblasts was as follows: a non-genic D4Z4-adjacent sequence (p13E-11, array-proximal)> untranscribed gene standards > D4Z4 arrays> constitutive heterochromatin (satellite 2; P < 10⁻⁴ for all comparisons). Cancers displaying D4Z4 hypermethylation also exhibited a hypermethylation-resistant subregion within the 3.3-kb D4Z4 repeat units. This subregion contains runs of G that form G-quadruplexes in vitro. Unusual DNA structures might contribute to topological constraints that link short 4q D4Z4 arrays to FSHD and make long ones phenotypically neutral.     

Bacteria-derived human growth hormone lacks lipolytic activity in rat adipose tissue

Bacteria-derived human growth hormone (hGH) shows little $\text{in}\text{vitro}$ lipolytic activity in adipose tissue from fed rats. In adipose tissue from fasted rats no lipolytic activity is observed. However, bacteria-derived hGH increased serum free fatty acids after intraperitoneal administration to hypophysectomized rats to the same extent as purified pituitary hGH. The dose response of the bacteria-derived hGH tested for $\text{in}\text{vitro}$ insulin-like activity was very similar to the pituitary extracted material. Thus bacteria-derived hGH behaves in a manner indistinguishable from highly purified preparations of pituitary hGH.