Estrogen-dependent expression of the chicken very low density apolipoprotein II gene in serum-free cultures of LMH cells

Summary The estrogen-responsive Leghorn strain M chicken hepatoma (LMH) cell line provides a model system for studying the estrogen-dependent, liver-specific expression of avian genes. Serum-free culture conditions have been established that allow expression of apolipoprotein B, very low density apolipoprotein II (apoVLDLII), serum albumin, and transferrin at levels detectable by Northern blot analysis. Regulation of apoVLDLII mRNA by estrogen occurred in an appropriate time-and dose-dependent manner in serum-free cultures of the LMH cells. The expression of apoVLDLII mRNA in serum-free culture was at least 100-fold higher than that expressed in cultures containing 10% serum. The level of estrogen receptors in LMH cells cultured with 10% serum was approximately 2000 receptors per cell, and in serum-free culture approximately 1000 receptors per cell. When these cells were transfected with estrogen receptor DNA and cultured in serum-free medium, apoVLDLII mRNA was decreased relative to that expressed in cells transfected with a control plasmid. These results indicate that when the LMH cells are cultured without serum, estrogen receptors are not the limiting factor for the expression of the apoVLDLII gene.     

Determinants of the DNA-binding specificity of the Avian homeodomain protein, AKR.

AKR (Avian Knotted-Related) was the first example of a vertebrate homeodomain protein with a highly divergent Ile residue at position 50 of the DNA-recognition helix. The protein was cloned from a liver cDNA expression library of a day-9 chick embryo by virtue of its ability to bind to the F' site in the proximal promoter of the avian apoVLDLII gene. Expression of the apoVLDLII gene is completely estrogen dependent, and mutation or deletion of the F' site decreases estrogen inducibility 5- to 10-fold. Subsequent data indicated that AKR is capable of repressing the hormone responsiveness of the apoVLDLII promoter, specifically through binding to F'. Involvement of the F' site in the hormone-dependent activation of apoVLDLII gene expression, as well as AKR-mediated repression, strongly suggests that both positive and negative regulatory factors interact with this site. Although several mammalian proteins have now been isolated whose homeodomains share many of the structural features of AKR, including the Ile at position 50, little is known of their functions in vivo or the identities of the genes they regulate. Consequently, the elements through which they exert their effects and the structural determinants of their binding specificities remain largely uncharacterized. In this study, we defined the sequence specificity of binding by AKR using polymerase chain reaction-assisted optimal site selection and determined the affinity with which the protein binds to both the optimized site and the F' site. Additionally, we generated a three-dimensional model of the AKR homeodomain binding to its optimized site and probed the validity of the model by examining the consequences of mutating amino acid residues in recognition helix 3 and the N-terminal arm on the binding specificity of the homeodomain. Finally, we present evidence that the F' site itself may act as an estrogen response element (ERE) when in the vicinity of imperfect or canonical EREs and that AKR can repress hormone inducibility mediated via this site.     

Conserved sequence motifs upstream from the co-ordinately expressed vitellogenin and apoVLDLII genes of chicken.

The vitellogenin and apoVLDLII yolk protein genes of chicken are transcribed in the liver upon estrogenization. To get information on putative regulatory elements, we compared more than 2 kb of their 5' flanking DNA sequences. Common sequence motifs were found in regions exhibiting estrogen-induced changes in chromatin structure. Stretches of alternating pyrimidines and purines of about 30-nucleotides long are present at roughly similar positions. A distinct box of sequence homology in the chicken genes also appears to be present at a similar position in front of the vitellogenin genes of Xenopus laevis, but is absent from the estrogen-responsive egg-white protein genes expressed in the oviduct. In front of the vitellogenin (position -595) and the VLDLII gene (position -548), a DNA element of about 300 base-pairs was found, which possesses structural characteristics of a mobile genetic element and bears homology to the transposon-like Vi element of Xenopus laevis.     

Characterization of liver-enriched proteins binding to a developmentally demethylated site flanking the avian apoVLDLII gene.

Although the avian apoVLDLII gene is normally expressed exclusively in the liver of the laying hen, the gene can be activated by estrogen in birds of either sex beginning between days 7-9 of embryogenesis. Developmentally programmed demethylation of sites in the 5'- and 3'-flanking regions of the gene have been shown to occur during this period of embryogenesis, suggesting that they may reflect changes in protein-DNA interactions that are involved in the acquisition of competence to activate the apoVLDLII gene. We have detected specific protein interactions at one location approximately 2.6 kb upstream from the apoVLDLII gene, that includes an Msp I site whose methylation status changes between days 7 and 9 of embryogenesis. The sequence of this region bears significant similarity to binding sites of members of the bZIP family of liver-enriched or -specific factors such as C/EBP, DBP, and LAP, that are characteristically produced relatively late during liver development. In the studies described here, we demonstrate that proteins binding to the upstream apoVLDLII site do not correspond to previously identified liver-enriched or -specific factors. They also display a pattern of activity during development and in human and avian hepatoma cell lines indicating that their expression is increased in proliferating cells. Southwestern blotting and UV cross-linking studies indicate that two proteins of approximately 60 kD are capable of binding to the site and we describe the purification of these factors from crude nuclear protein extracts obtained from rooster liver.     

Detection and characterization of degradative intermediates of avian apo very low density lipoprotein II mRNA present in estrogen-treated birds and following destabilization by hormone withdrawal

The apo very low density lipoprotein II (apoVLDLII) gene is dormant in embryos, chicks, and roosters but can be activated by estrogen. ApoVLDLII mRNA is relatively stable in estrogen-treated birds. However, its stability decreases 4-5-fold following withdrawal of hormone. We have characterized degradative intermediates of apoVLDLII mRNA detected in liver total RNA from estrogen-treated birds and searched for alterations in the pattern of intermediates that occur upon hormone-withdrawal. Primer extension and S1 nuclease analyses have demonstrated that these intermediates consist of fragments of the molecule with intact 5' ends but which lack various 3' regions. Estrogen withdrawal results in a decrease in the steady state levels of several of these intermediates and the detection of two new species. The end points of the major fragments present in RNA from both estrogen-treated and withdrawn birds all map in, or within four nucleotides of, the tetranucleotide, GAUG. The two fragments detected only in RNA from withdrawn birds have 3' ends that immediately precede the sequence, CAGU. Based on secondary structures predicted by a global folding program, the end points also appear to be preferentially located in, or at the base of, internal "bulge-loops".     

Cloning and characterization of a non-TIR-NBS-LRR type disease resistance gene analogue from peach.

Cloning of plant disease resistant genes is greatly helpful for disease resistant breeding in plants and the insight of resistance mechanism. However, there are less relevant researches in peach [prunus persica (L.) Batch]. In this study, four NBS-LRR type resistance gene analogs (RGAs) were cloned from genomic DNA of peach. The PNBS2 fragment was also amplified from peach cDNA and the full-length cDNA of PNBS2 (PRPM1, GenBank accession no. AY599223) has been cloned. Sequence analysis indicated that the cDNA of PRPM1 is 3007 bp in length and that the contained ORF encodes for a polypeptide of 917 amino acids. Compared with known NBS-LRR genes, it presented relatively high amino acid sequence identity. The polypeptide has typical structure of non-TIR-NBS-LRR genes, with NB-ARC, LZ, LRR and transmembrane domains. Southern analysis indicated that the PRPM1 gene might be a single copy in peach genome. Northern blot and RT-PCR analysis showed that the expression of PRPM1 was not induced by salicylic acid (SA) in peach young leaves. The isolation of putative resistance genes from peach provided useful bases for studying the structure and function of peach disease-resistance relating genes and disease resistant genetic breeding in peach.     

Analysis of the mouse gamma-crystallin gene family: assignment of multiple cDNAs to discrete genomic sequences and characterization of a representative gene.

Blot hybridization analysis of mouse DNA with gamma-crystallin-specific cDNAs has detected the presence of a multigene family comprised of at least four related genes. The detailed structure of one of these genes, mouse gamma 4-crystallin (M gamma 4.1), and its corresponding cDNA has been determined. The gene spans approximately 2.6 kilobases (kb) and contains two introns. The gene predicts a polypeptide of 174 amino acids that shares extensive sequence homology with gamma-crystallin polypeptides of other species. The two similar structural domains of the protein correspond exactly to the second and third exons of the gene, supporting an exon-duplication model of gene evolution. The similarity in structure of this gene to that recently reported for a gamma-crystallin gene of the rat (1) suggests that a common structure may exist for all gamma-crystallin genes of the two species. Moreover, a highly conserved region, 50 nucleotides in length, immediately precedes the TATA box of both the mouse and rat genes, suggesting that this sequence may be important in gene regulation.     

The chloroplast ribosomal intron of Chlamydomonas reinhardii codes for a polypeptide related to mitochondrial maturases.

The sequences of the 888bp chloroplast ribosomal intron and of the flanking 23S rRNA gene regions of Chlamydomonasreinhardii have been established. The intron can be folded with a secondary structure which is typical of group I introns of fungal mitochondrial genes. It contains a 489bp open reading frame encoding a potential polypeptide that is related to mitochondrial maturases.     

The bacteriophage T4 regA gene: primary sequence of a translational repressor

The regA gene product of bacteriophage T4 is an autogenously controlled translational regulatory protein that plays a role in differential inhibition (translational repression) of a subpopulation of T4-encoded "early" mRNA species. The structural gene for this polypeptide maps within a cluster of phage DNA replication genes, (genes 45-44-62-regA-43-42), all but one of which (gene 43) are under regA-mediated translational control. We have cloned the T4 regA gene, determined its nucleotide sequence, and identified the amino-terminal residues of a plasmid-encoded, hyperproduced regA protein. The results suggest that the T4 regA gene product is a 122 amino acid polypeptide that is mildly basic and hydrophilic in character; these features are consistent with known properties of regA protein derived from T4-infected cells. Computer-assisted analyses of the nucleotide sequences of the regA gene and its three upstream neighbors (genes 45, 44, and 62) suggest the existence of three translational initiation units in this four-gene cluster; one for gene 45, one for genes 44, 62 and regA, and one that serves only the regA gene. The analyses also suggest that the gene 44-62 translational unit harbors a stable RNA structure that obligates translational coupling of these two genes.     

The moxFG region encodes four polypeptides in the methanol-oxidizing bacterium Methylobacterium sp. strain AM1.

The polypeptides encoded by a putative methanol oxidation (mox) operon of Methylobacterium sp. strain AM1 were expressed in Escherichia coli, using a coupled in vivo T7 RNA polymerase/promoter gene expression system. Two mox genes had been previously mapped to this region: moxF, the gene encoding the methanol dehydrogenase (MeDH) polypeptide; and moxG, a gene believed to encode a soluble type c cytochrome, cytochrome cL. In this study, four polypeptides of Mr 60,000, 30,000, 20,000, and 12,000 were found to be encoded by the moxFG region and were tentatively designated moxF, -J, -G, and -I, respectively. The arrangement of the genes (5' to 3') was found to be moxFJGI. The identities of three of the four polypeptides were determined by protein immunoblot analysis. The product of moxF, the Mr-60,000 polypeptide, was confirmed to be the MeDH polypeptide. The product of moxG, the Mr-20,000 polypeptide, was identified as mature cytochrome cL, and the product of moxI, the Mr-12,000 polypeptide, was identified as a MeDH-associated polypeptide that copurifies with the holoenzyme. The identity of the Mr-30,000 polypeptide (the moxJ gene product) could not be determined. The function of the Mr-12,000 MeDH-associated polypeptide is not yet clear. However, it is not present in mutants that lack the Mr-60,000 MeDH subunit, and it appears that the stability of the MeDH-associated polypeptide is dependent on the presence of the Mr-60,000 MeDH polypeptide. Our data suggest that both the Mr-30,000 and -12,000 polypeptides are involved in methanol oxidation, which would bring to 12 the number of mox genes in Methylobacterium sp. strain AM1.     

Identification of a new gene in an operon for cellulose biosynthesis in Acetobacter xylinum

DNA sequencing of the region downstream of the cellulose synthase catalytic subunit gene of Acetobacter xylinum led to the identification of an open reading frame coding for a polypeptide of 86 kDa. The deduced amino acid sequence of this polypeptide matches from position 27 to 40 with the N-terminal amino acid sequence determined for a 93 kDa polypeptide that copurifies with the cellulose synthase catalytic subunit during purification of cellulose synthase. The cellulose synthase catalytic subunit gene and the gene encoding the 93 kDa polypeptide, along with other genes probably, are organized as an operon for cellulose biosynthesis in which the first gene is the catalytic subunit gene and the second gene codes for the 93 kDa polypeptide. The function of the 93 kDa polypeptide is not clear at present, however it appears to be tightly associated with the cellulose synthase catalytic subunit. Sequence analysis of the polypeptide shows that it is a membrane protein with a signal sequence at the N-terminal end and a transmembrane helix in the C-terminal region for anchoring it into the membrane.     

Mutations that detoxify an aberrant T4 membrane protein

We have investigated suppressors of the bacteriophage T4 rIIB toxic polypeptide encoded by the rIIB frameshift mutation FC238. We have found suppressors that eliminate the toxic polypeptide by creating new translational termination codons, that diminish the toxicity of the polypeptide by altering the amino acid sequence of the toxic protein, that alter the rIIA protein so as to influence toxicity, and that diminish the amount of toxic polypeptide by reducing the quantity of gene expression from the rIIB (FC238) gene. We propose that the toxicity of the FC238 polypeptide derives from its peculiar, bipartite structure and high membrane avidity. Suppressors that detoxify the FC238 polypeptide by missense probably disturb the bipartite structure and/or the affinity for the membrane. The distribution of transition mutations obtained with a variety of mutagens contributes to an appreciation of intrinsic mutability differences. Lastly, although suppressors of FC238 toxicity might emerge in phage genes other than rIIB and rIIA, none have been found.     

Nucleotide sequence of the Bacillus subtilis xylose isomerase gene: extensive homology between the Bacillus and Escherichia coli enzyme.

The xylose isomerase gene from Bacillus subtilis was cloned from a genomic BamH1 library by complementation of an isomerase defective Escherichia coli strain as previously described. The ATG initiation codon is preceded by a Shine-Dalgarno sequence and two hexamers being characteristic for the promoter region of Bacillus genes. The structural gene consists of 1320 base pairs, thus coding for a polypeptide chain of 440 amino acids with a molecular weight of 49 680. The polypeptide primary structure shows over 50% homology to that of the E. coli xylose isomerase.