The location of Z- and W-linked marker genes and sequence on the homomorphic sex chromosomes of the ostrich and the emu

Perhaps the most striking fact about early Cenozoic avian history some 70 million years ago was the rapid radiation of large, flightless, ground-living birds. It has been suggested that, for a time, there was active competition between these large terrestrial birds and the early mammals. Probably reflecting the above noted early start of Ratitae of the infraclass Eoaves, the presumptive sex chromosomes of their present day survivors, such as the emu and the ostrich, largely remained homomorphic. The signs of genetic differentiation between their still-homomorphic Z and W chromosomes were tested by using two marker genes (Z-linked ZOV3 and the gene for the iron-responsive element-binding protein) and one marker sequence of a part of a presumptive pseudogene (W-linked EE0.6 of the chicken). Their homologues, maintaining 71–92% identities to the chicken counterparts, were found in both the emu (Dromaius novaehollandiae) and the ostrich (Struthio camelus). Their locations were visualized on chromosome preparations by fluorescence in situ hybridization. In the case of the emu, these three marker sequences were localized on both members of the fifth pair of a female, thus revealing no sign yet of genetic differentiation between the Z and the W. The finding was the same with regard to both members of the fourth pair of male ostriches. In the female ostrich, however, the sequence of the gene for the iron-responsive element-binding protein was missing from one of the pairs, thus revealing the differentiation by a small deletion of the W from the Z.

trans/cis (Z/E) photoisomerization of the chromophore of photoactive yellow protein is not a prerequisite for the initiation of the photocycle of this photoreceptor protein

The chromophore of photoactive yellow protein (PYP) (i.e., 4-hydroxycinnamic acid) has been replaced by an analogue with a triple bond, rather than a double bond (by using 4-hydroxyphenylpropiolic acid in the reconstitution, yielding hybrid I) and by a “locked” chromophore (through reconstitution with 7-hydroxycoumarin-3-carboxylic acid, in which a covalent bridge is present across the vinyl bond, resulting in hybrid II). These hybrids absorb maximally at 464 and 443 nm, respectively, which indicates that in both hybrids the deprotonated chromophore does fit into the chromophore-binding pocket. Because the triple bond cannot undergo cis/trans (or E/Z) photoisomerization and because of the presence of the lock across the vinyl double bond in hybrid II, it was predicted that these two hybrids would not be able to photocycle. Surprisingly, both are able. We have demonstrated this ability by making use of transient absorption, low-temperature absorption, and Fourier-transform infrared (FTIR) spectroscopy. Both hybrids, upon photoexcitation, display authentic photocycle signals in terms of a red-shifted intermediate; hybrid I, in addition, goes through a blue-shifted-like intermediate state, with very slow kinetics. We interpret these results as further evidence that rotation of the carbonyl group of the thioester-linked chromophore of PYP, proposed in a previous FTIR study and visualized in recent time-resolved x-ray diffraction experiments, is of critical importance for photoactivation of PYP.

A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase

Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zα) that binds specifically to the left-handed Z-DNA conformation with high affinity (KD = 4 nM). As formation of Z-DNA in vivo occurs 5′ to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Zα has allowed identification of motifs common to this class of nucleic acid binding domain.

The solution structure of the Zα domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA

Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Zα. Here we report the solution structure of free Zα and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Zα)2/Z-DNA complex shows that most Z-DNA contacting residues in free Zα are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (α+β)helix–turn–helix/B-DNA complexes suggests that binding of Zα to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B- and Z-DNA.

Construction of a Z-DNA-specific restriction endonuclease

Novel restriction enzymes can be created by fusing the nuclease domain of FokI endonuclease with defined DNA binding domains. Recently, we have characterized a domain (Zα) from the N-terminal region of human double-stranded RNA adenosine deaminase (hADAR1), which binds the Z-conformation with high specificity. Here we report creation of a conformation-specific endonuclease, Zα nuclease, which is a chimera of Zα and FokI nuclease. Purified Zα nuclease cleaves negatively supercoiled plasmids only when they contain a Z-DNA forming insert, such as (dC-dG)13. The precise location of the cleavage sites was determined by primer extension. Cutting has been mapped to the edge of the B-Z junction, suggesting that Zα nuclease binds within the Z-DNA insert, but cleaves in the nearby B-DNA, by using a mechanism similar to type IIs restriction enzymes. These data show that Zα binds Z-DNA in an environment similar to that in a cell. Zα nuclease, a structure-specific restriction enzyme, may be a useful tool for further study of the biological role of Z-DNA.

Plasma Membrane Localization of Gαz Requires Two Signals

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the α subunits are myristoylated and/or palmitoylated, whereas the γ chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled α subunit, αz. Wild-type αz (αz-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does αz-WT. Removal of the myristoylation site produced a mutant αz that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of βγ with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on αz-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on αz-WT. Together these results suggest that myristate and βγ promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on αz specific localization at the plasma membrane.

Chemical chaperones mediate increased secretion of mutant α1-antitrypsin (α1-AT) Z: A potential pharmacological strategy for prevention of liver injury and emphysema in α1-AT deficiency

In α1-AT deficiency, a misfolded but functionally active mutant α1-ATZ (α1-ATZ) molecule is retained in the endoplasmic reticulum of liver cells rather than secreted into the blood and body fluids. Emphysema is thought to be caused by the lack of circulating α1-AT to inhibit neutrophil elastase in the lung. Liver injury is thought to be caused by the hepatotoxic effects of the retained α1-ATZ. In this study, we show that several “chemical chaperones,” which have been shown to reverse the cellular mislocalization or misfolding of other mutant plasma membrane, nuclear, and cytoplasmic proteins, mediate increased secretion of α1-ATZ. In particular, 4-phenylbutyric acid (PBA) mediated a marked increase in secretion of functionally active α1-ATZ in a model cell culture system. Moreover, oral administration of PBA was well tolerated by PiZ mice (transgenic for the human α1-ATZ gene) and consistently mediated an increase in blood levels of human α1-AT reaching 20–50% of the levels present in PiM mice and normal humans. Because clinical studies have suggested that only partial correction is needed for prevention of both liver and lung injury in α1-AT deficiency and PBA has been used safely in humans, it constitutes an excellent candidate for chemoprophylaxis of target organ injury in α1-AT deficiency.

Myozenin: An α-actinin- and γ-filamin-binding protein of skeletal muscle Z lines

To better understand the structure and function of Z lines, we used sarcomeric isoforms of α-actinin and γ-filamin to screen a human skeletal muscle cDNA library for interacting proteins by using the yeast two-hybrid system. Here we describe myozenin (MYOZ), an α-actinin- and γ-filamin-binding Z line protein expressed predominantly in skeletal muscle. Myozenin is predicted to be a 32-kDa, globular protein with a central glycine-rich domain flanked by α-helical regions with no strong homologies to any known genes. The MYOZ gene has six exons and maps to human chromosome 10q22.1-q22.2. Northern blot analysis demonstrated that this transcript is expressed primarily in skeletal muscle with significantly lower levels of expression in several other tissues. Antimyozenin antisera stain skeletal muscle in a sarcomeric pattern indistinguishable from that seen by using antibodies for α-actinin, and immunogold electron microscopy confirms localization specifically to Z lines. Thus, myozenin is a skeletal muscle Z line protein that may be a good candidate gene for limb-girdle muscular dystrophy or other neuromuscular disorders.

The universe at z > 5: When and how did the “dark age” end?

This paper considers how the first subgalactic structures produced the UV radiation that ionized the intergalactic medium before z = 5 and the “feedback” effects of the UV radiation on structure formation. The first “pregalaxies” may eventually be detectable by their direct UV emission, with characteristic spectral features at Lyman α; high-z supernovae may also be detectable. Other probes of the intergalactic medium beyond z = 5, and of the epochs of reheating and reionization, are discussed, along with possible links between the diffusion of pregalactic metals and the origin of magnetic fields.

Functional analysis of retinoid Z receptor beta, a brain-specific nuclear orphan receptor.

The retinoid Z receptor beta (RZR beta), an orphan receptor, is a member of the retinoic acid receptor (RAR)/thyroid hormone receptor (TR) subfamily of nuclear receptors. RZR beta exhibits a highly restricted brain-specific expression pattern. So far, no natural RZR beta target gene has been identified and the physiological role of the receptor in transcriptional regulation remains to be elucidated. Electrophoretic mobility shift assays reveal binding of RZR beta to monomeric response elements containing the sequence AnnTAGGTCA, but RZR beta-mediated transactivation of reporter genes is only achieved with two property spaced binding sites. We present evidence that RZR beta can function as a cell-type-specific transactivator. In neuronal cells, GaI-RZR beta fusion proteins function as potent transcriptional activators, whereas no transactivation can be observed in nonneuronal cells. Mutational analyses demonstrate that the activation domain (AF-2) of RZR beta and RAR alpha are functionally interchangeable. However, in contrast to RAR and TR, the RZR beta AF-2 cannot function autonomously as a transactivation domain. Furthermore, our data define a novel repressor function for the C-terminal part of the putative ligand binding domain. We propose that the transcriptional activity of RZR beta is regulated by an interplay of different receptor domains with coactivators and corepressors.

Chromosomal localization of the proteasome Z subunit gene reveals an ancient chromosomal duplication involving the major histocompatibility complex.

Proteasomes are the multi-subunit protease thought to play a key role in the generation of peptides presented by major histocompatibility complex (MHC) class I molecules. When cells are stimulated with interferon gamma, two MHC-encoded subunits, low molecular mass polypeptide (LMP) 2 and LMP7, and the MECL1 subunit encoded outside the MHC are incorporated into the proteasomal complex, presumably by displacing the housekeeping subunits designated Y, X, and Z, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably by altering the cleavage specificities of the proteasome. Here we show that the mouse gene encoding the Z subunit (Psmb7) maps to the paracentromeric region of chromosome 2. Inspection of the mouse loci adjacent to the Psmb7 locus provides evidence that the paracentromeric region of chromosome 2 and the MHC region on chromosome 17 most likely arose as a result of a duplication that took place at an early stage of vertebrate evolution. The traces of this duplication are also evident in the homologous human chromosome regions (6p21.3 and 9q33-q34). These observations have implications in understanding the genomic organization of the present-day MHC and offer insights into the origin of the MHC.

Transcription of the human corticotropin-releasing hormone gene in NPLC cells is correlated with Z-DNA formation.

The intron of the corticotropin-releasing hormone (corticoliberin; CRH) gene contains a sequence of over 100 bp of alternating purine/pyrimidine residues. We have used binding of a Z-DNA-specific antibody in metabolically active, permeabilized nuclei to study the formation of Z-DNA in this sequence at various levels of transcription. In the NPLC human primary liver carcinoma cell line, activation of cAMP-dependent pathways increased the level of transcription, while adding glucocorticoids inhibited transcription of the CRH gene. These cells respond in a manner similar to hypothalamic cells. Z-DNA formation in this sequence was detected at the basal level of transcription, as well as after stimulation with forskolin. Inhibition of transcription by dexamethasone abolished Z-DNA formation. Z-DNA formation in the WC gene (c-myc) was affected differently in the same experiment. Thus, changes in Z-DNA formation in the CRH gene are gene specific and are linked to the transcription of the gene.

Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins.

We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.

Z-DNA-forming sites within the human beta-globin gene cluster.

Agarose-encapsulated, metabolically active, permeabilized nuclei from human hematopoietic cell lines were tested for Z-DNA formation in the beta-globin gene cluster. Biotinylated monoclonal antibodies against Z-DNA were diffused into the nuclei and cross-linked to DNA with a 10-ns laser exposure at 266 nm. Following digestion with restriction enzymes, fragments that had formed Z-DNA were isolated. Seventeen regions with Z-DNA sequence motifs in the 73-kb region were studied by PCR amplification, and five were found in the Z conformation.

Chicken double-stranded RNA adenosine deaminase has apparent specificity for Z-DNA.

A M(r) 140,000 protein has been purified from chicken lungs to apparent homogeneity. The protein binds with high affinity to a non-BNA conformation, which is most likely to the Z-DNA. The protein also has a binding site for double-stranded RNA (dsRNA). Peptide sequences from this protein show similarity to dsRNA adenosine deaminase, an enzyme that deaminates adenosine in dsRNA to form inosine. Assays for this enzyme confirm that dsRNA adenosine deaminase activity and Z-DNA binding are properties of the same molecule. The coupling of these two activities in a single molecule may indicate a distinctive mechanism of gene regulation that is, in part, dependent on DNA topology. As such, DNA topology, through its effects on the efficiency and extent of RNA editing may be important in the generation of new phenotypes during evolution.