Evidence that the hormone-binding domain of the mouse glucocorticoid receptor directly represses DNA binding activity in a major portion of receptors that are "misfolded" after removal of hsp90.

After dissociation of cytosolic heteromeric glucocorticoid receptor complexes by steroid, salt, and other methods, only 35-60% of the dissociated receptors can bind to DNA-cellulose. The DNA-binding and non-DNA-binding forms of the dissociated receptors have the same Mr and are phosphorylated to the same extent (Tienrungroj, W., Sanchez, E. R., Housley, P. R., Harrison, R. W., and Pratt, W. B. (1987) J. Biol. Chem. 262, 17347-17349). The basis for the different DNA-binding activities is unknown, but the DNA-binding fraction of the receptor has a more basic pI than the non-DNA-binding fraction (Smith, A. C., Elsasser, M. S., and Harmon, J. M. (1986) J. Biol. Chem. 261, 13285-13292). We have separated the non-DNA-binding state of the receptor from the DNA-binding state and then cleaved it with trypsin and chymotrypsin. We find that the 15-kDa tryptic fragment derived from the non-DNA-binding state of the dissociated receptor is fully competent in binding DNA, whereas the 42-kDa chymotryptic fragment containing both the hormone-binding and DNA-binding domains does not bind DNA. Trypsin cleavage of the molybdate-stabilized untransformed receptor also yields a 15-kDa fragment that is fully competent in binding DNA. Reducing agents do not restore DNA-binding to the non-DNA-binding fraction of the receptor and the hormone-binding domain can be separated from the DNA-binding domain on nonreducing gel electrophoresis. These results argue that the two domains are not linked by disulfide bridges, and they are consistent with the proposal that there are two least energy states of folding after dissociation of hsp90. A significant portion of the receptors is "misfolded" in such a manner that the steroid binding domain is directly preventing DNA-binding activity.     

Master genes in mammalian repetitive DNA amplification.

The analysis of species-specific subfamilies of both the LINE and SINE mammalian repetitive DNA families suggests that such subfamilies have arisen by amplification of an extremely small group of 'master' genes. In contrast to the master genes, the vast majority of both SINEs and LINEs appear to behave like psudogenes in their inability to undergo extensive amplification.     

The effect of cessation of growth on protein synthesis in a mutant of Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase

A temperature-sensitive mutant of Chinese hamster ovary cells with an altered leucyl-tRNA synthetase fails to grow and to incorporate amino acids into protein properly at or near the non-permissive temperature. This mutant was used to determine whether cessation of growth at the elevated temperature affected elongation factor EF-1, since the activity of EF-1 is markedly lower in non-growing cells in stationary phase than in rapidly-growing cells in exponential phase. Cell-free extracts prepared from cells maintained at 39°C for 24 h showed a marked decrease in the ability to translate natural mRNAs, compared to cells incubated at 34°C. However, the ability to translate poly(U), which requires elongation factor EF-1 (and EF-2), was not affected. Analyses of activities involved in the initiation of protein synthesis and in the activation of amino acids revealed that, with the exception of leucyl-tRNA synthetase, the rest of the components required for translation also appeared to be relatively stable even after 24 h at the elevated temperature. The effects of elevated temperature on cell-free extracts were also investigated. The results were similar to those obtained with intact cells; that is, except for leucyl-tRNA synthetase which was rapidly inactivated in vitro at 39°C, other aminoacyl-tRNA synthetases and translational components involved in chain initiation and elongation were relatively stable. Thus, no change in EF-1 activity was detected as a result of arrested cell growth, an inherent lability of the elongation factor, or metabolic degradation as a consequence of a rapid turnover rate in the absence of protein synthesis.     

The effect of elevated temperature on protein synthesis in cell-free extracts of cultured Chinese hamster ovary cells

The effect of elevated temperature on the activity of various components involved in protein synthesis was investigated in extracts from cultured Chinese hamster ovary cells. The translation of exogenous mRNA was markedly inhibited by preincubation of the extract for 15 to 20 minutes at 42°C. However, the following intermediary reactions were not affected, or only slightly inhibited, at 42°C: 1) the incorporation of Met-tRNA_f into eIF-2·Met-tRNA_f·GTP ternary complex; 2) the interaction of the ternary complex with 40S ribosomal subunits to form the 40S preinitiation intermediate; 3) the binding of mRNA and 60S subunits to form the 80S initiation complex; and 4) the reactions catalyzed by elongation factors EF-1 and EF-2. The activity of Met-tRNA synthetase was markedly inhibited, affecting the formation of initiator Met-tRNA_f required for the initiation of protein synthesis and the translation of natural mRNA. Other aminoacyl-tRNA synthetases were not significantly affected by the elevated temperature.     

Ultrastructural studies on the sporulation of oocysts of Toxoplasma gondii. II. Formation of the sporocyst and structure of the sporocyst wall

The ultrastructural changes observed during sporocyst formation and the structure of the sporocyst wall was examined in oocysts which had been allowed to sporulate for between 12 and 48 hours at 27 degrees C. As the spherical sporoblast developed into the sporocyst the cytoplasmic mass became ellipsoidal in shape although no change was noted in the organelle compliment, which cosisted of two nuclei plus a number of polysaccharide granules, lipid globules, mitochondria, Golgi bodies, and some rough endoplasmic reticulum. The sporocyst wall consisted of a thin outer layer (15-20 nm) which was formed from two limiting membranes of the sporoblast and an inner layer (40-50 nm) which was comprised of four curved plates. This inner layer was formed under the outer layer and, although no specific cytoplasmic organelle disappeared with its formation, some unit membranes were observed close to the plasmalemma during its formation. Each curved plate has a marginal swelling and an interposing strip of material is present between the margins of adjacent plates. The plates are joined to the interposing strip by a thin band of osmiophilic material. In oblique and tangential sections through the plates two types of cross banding were observed which differed in periodicity.     

The mitotic chromosomes of Notophthalmus (=Triturus) viridescens: Localization of C banding regions and DNA sequences complementary to 18S, 28S and 5S ribosomal RNA

The metaphase chromosomes of Notophthalmus (Triturus) viridescens have been studied by C-banding and in situ hybridization. The chromosomes show the pericentric C-banding seen in many organisms and in addition have interstitial C-bands located a short distance from the pericentric C-bands on each chromosome arm. A few C-bands are seen in telomeric regions. Regions which hybridize in situ with 18S and 28S ribosomal RNA were found on three chromosome pairs. The animals studied fell into three groups with respect to which of the six possible sites showed detectable hybridization with 18S and 28S RNA. Individual animals differed not only in the pattern of in situ hybridization of ribosomal RNA but also in the number of ribosomal RNA cistrons in the genome as measured by saturation hybridization on purified DNA. In situ hybridization showed five pairs of chromosomes which contained DNA complementary to 5S RNA. The four pairs of subtelocentric chromosomes in the N. viridescens karyotype all have 5S DNA in the pericentric regions. The fifth cluster of 5S DNA is in the middle of one arm of the chromosomes in one of the two smallest submetacentric pairs in the genome. The five sites of 5S DNA differ markedly in the level of in situ hybridization with 5S cRNA.     

On the information content of the genetic code

In living organisms 20 amino acids along with the terminator value(s) are encoded by 64 codons giving a degeneracy of the codons as described by the genetic code. A basic theoretical problem of genetic codes is to explain the particular distribution of degeneracies of partitions involved in the codes. In this work the degeneracy problem is considered in the framework of information theory. It is shown by direct numerical evaluation of a certain degeneracy information function associated with the genetic code that the degeneracy of the codes is observed to be related to the optimization of this function.     

Proteomic Insights into Endometrial Receptivity and Embryo‐Endometrial Epithelium Interaction for Implantation Reveal Critical Determinants of Fertility

In vitro fertilization has overcome infertility issues for many couples. However, achieving implantation of a viable embryo into the maternal endometrium remains a limiting step in optimizing pregnancy success. The molecular mechanisms which characterize the transient state of endometrial receptivity, critical in enabling embryo‐endometrial interactions, and proteins which underpin adhesion at the implantation interface, are limited in humans despite these temporally regulated processes fundamental to life. Hence, failure of implantation remains the “final frontier” in infertility. A human coculture model is utilized utilizing spheroids of a trophectoderm (trophoblast stem) cell line, derived from pre‐implantation human embryos, and primary human endometrial epithelial cells, to functionally identify “fertile” versus “infertile” endometrial epithelium based on adhesion between these cell types. Quantitative proteomics identified proteins associated with human endometrial epithelial receptivity (“epithelial receptome”) and trophectoderm adhesion (“adhesome”). As validation, key “epithelial receptome” proteins (MAGT‐1/CDA/LGMN/KYNU/PC4) localized to the epithelium of receptive phase (mid‐secretory) endometrium obtained from fertile, normally cycling women but is largely absent from non‐receptive (proliferative) phase tissues. Factors involved in embryo‐epithelium interaction in successive temporal stages of endometrial receptivity and implantation are demonstrated and potential targets for improving fertility are provided, enhancing potential to become pregnant either naturally or in a clinical setting.