bluuebunny/crossref_metadata_2025 huggingface dataset:
Negative regulation of globin gene expression during megakaryocytic differentiation of a human erythroleukemic cell line.
Authors: Lumelsky N L , Forget B G
Published: October 2015

The human erythroleukemic cell line K562 was used as a model for analysis of the mechanisms responsible for alterations in gene expression during differentiation. K562 cells normally synthesize fetal hemoglobin (gamma-globin), but treatment with tumor-promoting phorbol esters (phorbol myristate acetate and tetradecanoyl phorbol acetate) results in the loss of the erythroid phenotype of the cells and causes a shift toward a megakaryocytic phenotype. This shift involves markedly decreased production of fetal hemoglobin and de novo synthesis of a number of proteins specific for megakaryocytes. The results of this work indicate that negative regulation of fetal hemoglobin during megakaryocytic differentiation of K562 cells occurs at the level of down regulation of gamma-globin mRNA accumulation. This effect consists of at least two components: reduction in the rate of transcription of the gamma-globin gene and decrease in stability of the normally very stable gamma-globin mRNA. We have developed two assay systems that permit investigation of the transcriptional and posttranscriptional effects of phorbol myristate acetate independently from each other. These assay systems make use of a heterologous reporter gene for the transcriptional analysis and a marked gamma-globin gene for the analysis of mRNA stability. The DNA sequences located in the 3' flanking region of the A gamma-globin gene were found to be responsible for the decrease in transcription rate.
In vivo characterization of the Saccharomyces cerevisiae centromere DNA element I, a binding site for the helix-loop-helix protein CPF1.
Authors: Niedenthal R , Stoll R , Hegemann J H
Published: October 2015

The centromere DNA element I (CDEI) is an important component of Saccharomyces cerevisiae centromere DNA and carries the palindromic sequence CACRTG (R = purine) as a characteristic feature. In vivo, CDEI is bound by the helix-loop-helix protein CPF1. This article describes the in vivo analysis of all single-base-pair substitutions in CDEI in the centromere of an artificial chromosome and demonstrates the importance of the palindromic sequence for faithful chromosome segregation, supporting the notion that CPF1 binds as a dimer to this binding site. Mutational analysis of two conserved base pairs on the left and two nonconserved base pairs on the right of the CDEI palindrome revealed that these are also relevant for mitotic CEN function. Symmetrical mutations in either half-site of the palindrome affect centromere activity to a different extent, indicating nonidentical sequence requirements for binding by the CPF1 homodimer. Analysis of double point mutations in CDEI and in CDEIII, an additional centromere element, indicate synergistic effects between the DNA-protein complexes at these sites.
byr2, a Schizosaccharomyces pombe gene encoding a protein kinase capable of partial suppression of the ras1 mutant phenotype.
Authors: Wang Y , Xu H P , Riggs M , Rodgers L , Wigler M
Published: October 2015

Schizosaccharomyces pombe contains a single gene, ras1, which is a homolog of the mammalian RAS genes. ras1 is required for conjugation, sporulation, and normal cell shape. ras1 has been previously identified as ste5. We report here a gene we call byr2 that can encode a predicted protein kinase and can partially suppress defects in ras1 mutants. ras1 mutant strains expressing high levels of byr2 can sporulate competently but are still defective in conjugation and abnormally round. byr2 mutants are viable and have normal shape but are absolutely defective in conjugation and sporulation. byr2 is probably identical to ste8. In many respects, byr2 resembles the byr1 gene, another suppressor of the ras1 mutation, which has been identified previously as ste1. Our data indicate that if ras1, byr2, and byr1 act along the same pathway, then the site of action for byr2 is between the sites for ras1 and byr1.
DNA-binding specificity of the fushi tarazu homeodomain.
Authors: Florence B , Handrow R , Laughon A
Published: October 2015

The fushi tarazu (ftz) gene of Drosophila melanogaster encodes a homeodomain-containing transcription factor that functions in the formation of body segments. Here we report an analysis of the DNA-binding properties of the ftz homeodomain in vitro. We provide evidence that the homeodomain binds to DNA as a monomer, with an equilibrium dissociation constant of 2.5 x 10(-11) M for binding to a consensus binding site. A single ftz binding site occupies 10 to 12 bp, as judged by the ability of protein bound at one site to interfere with binding to an adjacent site. These experiments also demonstrated a lack of cooperative binding between ftz homeodomains. Analysis of single-nucleotide substitutions over an 11-bp sequence shows that a stretch of 6 bp is critical for binding, with an optimal sequence of 5'CTAATTA3'. These data correlate well with recent structural evidence for base-specific contact at these positions. In addition, we found that sequences flanking the region of direct contact have effects on DNA binding that could be of biological significance.
Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.
Authors: Chakraborty T , Brennan T J , Li L , Edmondson D , Olson E N
Published: October 2015

Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenin's weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.
Ecdysterone receptor is a sequence-specific transcription factor involved in the developmental regulation of heat shock genes.
Authors: Luo Y , Amin J , Voellmy R
Published: October 2015

Purification of ecdysterone receptor from Drosophila melanogaster to apparent homogeneity is reported. Purified receptor binds specifically to several sequences in the promoters of the developmentally active hsp27 and hsp23 heat shock genes that were previously implied in ecdysterone regulation of the genes and that share limited homology among themselves and with mammalian steroid receptor binding sites. Some of these elements confer ecdysterone regulation on a basal promoter in transfected cells, acting in a synergistic fashion. Transcription in vitro of promoters containing such elements is stimulated up to 100-fold by added purified ecdysterone receptor, depending on receptor dosage and the number of elements present. Transcriptional enhancement requires sequence-specific binding of receptor to template promoters which facilitates the formation of a preinitiation complex. Ecdysterone stimulates DNA binding of the receptor in vitro.
Depletion of c-myc with specific antisense sequences reverses the transformed phenotype in ras oncogene-transformed NIH 3T3 cells.
Authors: Sklar M D , Thompson E , Welsh M J , Liebert M , Harney J , Grossman H B , Smith M , Prochownik E V
Published: October 2015

ras oncogene-transformed NIH 3T3 cells expressing glucocorticoid-inducible antisense c-myc cDNA transcripts at levels sufficient to deplete c-myc protein lost their transformed morphology and the ability to grow in soft agar; their ability to form tumors in nude mice was also impaired. These changes were dependent on the continuous expression of the antisense sequences. No major effects on plating efficiencies, growth rates in monolayer culture, or immortalization were observed in the revertant cells, indicating that the observed effects were not a toxic consequence of c-myc protein depletion. Transfection with the same vector expressing c-myc in the sense orientation or other control vectors had no effect on transformation. These results suggest that a certain minimum level of expression of c-myc is required for the maintenance of ras transformation in NIH 3T3 cells.
Changes in dihydrofolate reductase (DHFR) mRNA levels can account fully for changes in DHFR synthesis rates during terminal differentiation in a highly amplified myogenic cell line.
Authors: Schmidt E E , Merrill G F
Published: October 2015

Dihydrofolate reductase (DHFR) enzyme is preferentially synthesized in proliferative cells. A mouse muscle cell line resistant to 300 microM methotrexate was developed to investigate the molecular levels at which DHFR is down-regulated during myogenic withdrawal from the cell cycle. H- alpha R300T cells contained 540 copies of the endogenous DHFR gene and overexpressed DHFR mRNA and DHFR protein. Despite DHFR gene amplification, the cells remained diploid. As H- alpha R300T myoblasts withdrew from the cell cycle and committed to terminal differentiation, DHFR mRNA levels and DHFR synthesis rates decreased with closely matched kinetics. After 15 to 24 h, committed cells contained 5% the proliferative level of DHFR mRNA (80 molecules per committed cell) and synthesized DHFR protein at 6% the proliferative rate. At no point during the commitment process did the decrease in DHFR synthesis rate exceed the decrease in DHFR message. The decrease in DHFR mRNA levels during commitment was sufficient to account fully for the decrease in rates of DHFR synthesis. Furthermore, DHFR mRNA remained polysomal, and the average number of ribosomes per message remained constant (five to six ribosomes per DHFR mRNA). The constancy of polysome size, along with the uniform rate of DHFR synthesis per message, indicated that DHFR mRNA was efficiently translated in postreplicative cells. The results support a model wherein replication-dependent changes in DHFR synthesis rates are determined exclusively by changes in DHFR mRNA levels.
AAR1/TUP1 protein, with a structure similar to that of the beta subunit of G proteins, is required for a1-alpha 2 and alpha 2 repression in cell type control of Saccharomyces cerevisiae.
Authors: Mukai Y , Harashima S , Oshima Y
Published: October 2015

We have cloned a DNA fragment complementing the aar1 mutation defective in the a1-alpha 2 repression of the alpha 1 cistron and haploid-specific genes in Saccharomyces cerevisiae. Nucleotide sequence and mapping data indicated that the AAR1 gene is identical with TUP1, which is allelic to the SFL2, FLK1, CYC9, UMR7, AMM1, and AER2 genes, whose mutations are known to confer a variety of phenotypes, such as thymidine uptake, flocculation, insensitivity to glucose repression, a defect in UV-induced mutagenesis, and a defect in ARS plasmid maintenance. The TUP1/AER2 protein is known to have significant similarity with the beta subunits of G proteins in the C-terminal half, in two glutamine-rich domains in the N-terminal half, and in a central region rich in serine and threonine residues. Disruption of the chromosomal AAR1 gene in alpha and a/alpha cells conferred the nonmating phenotype, and the a/alpha diploids could not sporulate. The AAR1/TUP1 gene is transcribed into a 2.5-kb mRNA independently of the mating-type information of the cell. These observations and mRNA analysis of cell-type-specific genes indicated that the AAR1/TUP1 protein is also indispensable for a1-alpha 2 repression of RME1 and for alpha 2 repression of a-specific genes.
Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2.
Authors: Gregory R J , Rich D P , Cheng S H , Souza D W , Paul S , Manavalan P , Anderson M P , Welsh M J , Smith A E
Published: October 2015

One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP-stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients.
The B-cell and neuronal forms of the octamer-binding protein Oct-2 differ in DNA-binding specificity and functional activity.
Authors: Dent C L , Lillycrop K A , Estridge J K , Thomas N S , Latchman D S
Published: October 2015

B lymphocytes contain an octamer-binding transcription factor, Oct-2, that is absent in most other cell types and plays a critical role in the B-cell-specific transcription of the immunoglobulin genes. A neuronal form of this protein has also been detected in brain and neuronal cell lines by using a DNA mobility shift assay, and an Oct-2 mRNA is observed in these cells by Northern (RNA) blotting and in situ hybridization. We show that the neuronal form of Oct-2 differs from that found in B cells with respect to both DNA-binding specificity and functional activity. In particular, whereas the B-cell protein activates octamer-containing promoters, the neuronal protein inhibits octamer-mediated gene expression. The possible role of the neuronal form of Oct-2 in the regulation of neuronal gene expression and its relationship to B-cell Oct-2 are discussed.
A novel Ca2+/calmodulin-dependent protein kinase and a male germ cell-specific calmodulin-binding protein are derived from the same gene.
Authors: Means A R , Cruzalegui F , LeMagueresse B , Needleman D S , Slaughter G R , Ono T
Published: October 2015

A cDNA representing a unique Ca2+/calmodulin-dependent protein kinase has been cloned and sequenced from a rat brain cDNA library. This enzyme, expressed in brain, testis, and spleen, is only 32% identical to the various isoforms of Ca2+/calmodulin-dependent protein kinase II. The sequence of the COOH-terminal 169 amino acids is identical to that of a previously described male germ cell-specific calmodulin-binding protein called calspermin (T. Ono, G.R. Slaughter, R.G. Cook, and A.R. Means, J. Biol. Chem. 264:2081-2087, 1989). This identity extends to the nucleic acid sequence and includes all but the first 130 nucleotides of the calspermin cDNA. Primer extension and sequence of a genomic fragment containing the unique calspermin sequence reveals that this mRNA is derived from the kinase transcription unit by germ cell-specific use of a unique exon. In situ hybridization was used to demonstrate that both kinase and calspermin mRNAs are expressed during spermatogenesis. The kinase mRNA is first detected in early meiotic cells and declines to a low level in haploid cells. Calspermin mRNA first appears in pachytene primary spermatocytes and continues to increase as cells complete meiosis and undergo terminal differentiation. These results show that differential utilization of a single gene during spermatogenesis is used to generate mRNAs that encode proteins with distinct functions.
Protein truncation is required for the activation of the c-myb proto-oncogene.
Authors: Grässer F A , Graf T , Lipsick J S
Published: October 2015

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.
The Neurospora crassa cyt-20 gene encodes cytosolic and mitochondrial valyl-tRNA synthetases and may have a second function in addition to protein synthesis.
Authors: Kubelik A R , Turcq B , Lambowitz A M
Published: October 2015

The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A. Collins, H. Bertrand, R.J. LaPolla, and A.M. Lambowitz, Mol. Gen. Genet. 177:73-84, 1979). We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs). A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis. The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S. Kappy and R.L. Metzenberg, J. Bacteriol. 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene. The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants. The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant. The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth. The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis.
HSP47: a tissue-specific, transformation-sensitive, collagen-binding heat shock protein of chicken embryo fibroblasts.
Authors: Hirayoshi K , Kudo H , Takechi H , Nakai A , Iwamatsu A , Yamada K M , Nagata K
Published: October 2015

We report the isolation and characterization of a cDNA clone encoding HSP47, a transformation-sensitive heat shock protein that binds to collagen. A cDNA library was prepared from total RNA isolated from heat-shocked chicken embryo fibroblasts and screened by using oligonucleotide mixtures prepared on the basis of the N-terminal amino acid sequence of biochemically purified HSP47. The cDNA insert contained 3,278 bp, which encoded a 15-amino-acid signal peptide and a mature protein coding region consisting of 390 amino acid residues; it also included part of the 5' noncoding region and a long 3' noncoding region. The deduced amino acid sequence revealed an RDEL sequence at the C terminus, which is a variant of the KDEL retention signal for retention of proteins in the endoplasmic reticulum. Northern (RNA) blot analyses and nuclear run-on assays established that the induction of HSP47 by heat shock and its suppression after transformation of chicken embryo fibroblasts by Rous sarcoma virus are regulated at the transcriptional level. A homology search revealed that this protein belongs to the serpin family, the superfamily of plasma serine protease inhibitors. Although structurally homologous to the serpins, HSP47 lacks the active site thought to be essential for the inhibition of proteases and does not appear to bind to intracellular proteases. HSP47 is the first heat shock protein found to be a member of the serpin superfamily. Conversely, it is the first serpin family member that is not secreted from cells, which could be explained by acquisition of the RDEL retention signal during evolution.
The highly conserved N-terminal domains of histones H3 and H4 are required for normal cell cycle progression.
Authors: Morgan B A , Mittman B A , Smith M M
Published: October 2015

The N-terminal domains of the histones H3 and H4 are highly conserved throughout evolution. Mutant alleles deleted for these N-terminal domains were constructed in vitro and examined for function in vivo in Saccharomyces cerevisiae. Cells containing a single deletion allele of either histone H3 or histone H4 were viable. Deletion of the N-terminal domain of histone H4 caused cells to become sterile and temperature sensitive for growth. The normal cell cycle progression of these cells was also altered, as revealed by a major delay in progression through the G2 + M periods. Deletion of the N-terminal domain of histone H3 had only minor effects on mating and the temperature-sensitive growth of mutant cells. However, like the H4 mutant, the H3 mutants had a significant delay in completing the G2 + M periods of the division cycle. Double mutants containing N-terminal domain deletions of both histone H3 and histone H4 were inviable. The phenotypes of cells subject to this synthetic lethality suggest that the N-terminal domains are required for functions essential throughout the cell division cycle and provide genetic evidence that histones are randomly distributed during chromosome replication.
SHI, a new yeast gene affecting the spacing between TATA and transcription initiation sites.
Authors: Furter-Graves E M , Furter R , Hall B D
Published: October 2015

In a genetic selection for Saccharomyces cerevisiae genes involved in transcription start site specification, two mutant genes which restore alcohol dehydrogenase activity to a functionally defective S. pombe ADH gene were recovered. Examination of S. pombe ADH initiation sites showed that mutations in the SHI gene shift the location of the transcription initiation window closer to TATA. The shi mutant also affected initiation site selection for two S. cerevisiae genes that were tested. For H2B mRNA, initiation occurred in the shi mutant at a series of initiation sites located 43 to 80 bp 3' of the histone H2B TATA sequence and at the usual initiation sites 102 and 103 bp downstream of the TATA sequence. Weakly used initiation sites ranging from 51 to 80 bp downstream of the TATA sequence were observed for the S. cerevisiae ADH1 gene in shi strains, in addition to the normal ADH1 initiation sites 89 and 99 bp from the TATA sequence. Restoration of function to the defective S. pombe ADH gene occurs only when this gene contains a TATA sequence; a single-base-pair TATA-to-TAGA change is sufficient to prevent this restoration of function. Genetic mapping placed the SHI locus on the left arm of chromosome VII, 22.3 centimorgans from cyh2; it does not correspond to any previously mapped gene.
Functional characterization of the human hypoxanthine phosphoribosyltransferase gene promoter: evidence for a negative regulatory element.
Authors: Rincón-Limas D E , Krueger D A , Patel P I
Published: October 2015

The enzyme hypoxanthine phosphoribosyltransferase (HPRT) catalyzes the metabolic salvage of the purine bases hypoxanthine and guanine. We previously characterized the genomic structure of the human HPRT gene and described its promoter sequence. In this report, we identify cis-acting transcriptional control regions of the human HPRT gene by linking various 5'-flanking sequences to the bacterial chloramphenicol acetyltransferase gene. The sequence from positions -219 to -122 relative to the translation initiation site is required for maximal expression of this gene, and it functions equally in both normal and reverse orientations. In addition, a cis-acting negative element is present in the region spanning from positions -570 to -388. This negative element can also repress promoters of heterologous genes, such as those of adenosine deaminase and dihydrofolate reductase, which are structurally and functionally similar to the human HPRT promoter. Furthermore, this repressor element functions independently of its orientation but appears to be distance dependent. In vivo competition assays demonstrated that the trans-acting factor(s) that binds to this negative element specifically inhibits human HPRT promoter activity. Taken together, these data localize cis-acting sequences important in the regulation of human HPRT gene expression and should allow the study of protein-DNA interactions which modulate the transcription of this gene.
The AGA1 product is involved in cell surface attachment of the Saccharomyces cerevisiae cell adhesion glycoprotein a-agglutinin.
Authors: Roy A , Lu C F , Marykwas D L , Lipke P N , Kurjan J
Published: October 2015

Saccharomyces cerevisiae a and alpha cells express the complementary cell surface glycoproteins a-agglutinin and alpha-agglutinin, respectively, which interact with one another to promote cellular aggregation during mating. Treatment of S. cerevisiae a cells with reducing agents releases the binding subunit of a-agglutinin, which has been purified and characterized; little biochemical information on the overall structure of a-agglutinin is available. To characterise a-agglutinin structure and function, we have used a genetic approach to clone an a-agglutinin structural gene (AGAI). Mutants with a-specific agglutination defects were isolated, the majority of which fell into a single complementation group, called aga1. The aga1 mutants showed wild-type pheromone production and response, efficient mating on solid medium, and a mating defect in liquid medium; these phenotypes are characteristic of agglutinin mutants. The AGA1 gene was cloned by complementation; the gene sequence indicated that it could encode a protein of 725 amino acids with high serine and threonine content, a putative N-terminal signal sequence, and a C-terminal hydrophobic sequence similar to signals for the attachment to glycosyl phosphatidylinositol anchors. Active a-agglutinin binding subunit is secreted by aga1 mutants, indicating that AGA1 is involved in cells surface attachment of a-agglutinin. This result suggests that AGA1 encodes a protein with functional similarity to the core subunits of a-agglutinin analogs from other budding yeasts. Unexpectedly, the AGA1 transcript was expressed and induced by pheromone in both a and alpha cells, suggesting that the a-specific expression of active a-agglutinin results only from a-specific regulation of the a-agglutinin binding subunit.
Cloning and characterization of a gene which determines osmotic stability in Saccharomyces cerevisiae.
Authors: Stateva L I , Oliver S G , Trueman L J , Venkov P V
Published: October 2015

The srb1-1 mutation of Saccharomyces cerevisiae is an ochre allele which renders the yeast dependent on an osmotic stabilizer for growth and gives the cells the ability to lyse on transfer to hypotonic conditions. A DNA fragment which complements both of these phenotypic effects has been cloned. This clone contains a functional gene which is transcribed into a 2.3-kb polyadenylated mRNA molecule. Transformation of yeast strains carrying defined suppressible alleles demonstrated that the cloned fragment does not contain a nonsense suppressor. Integrative transformation and gene disruption experiments, when combined with classical genetic analysis, confirmed that the cloned fragment contained the wild-type SRB1 gene. The integrated marker was used to map SRB1 to chromosome XV by Southern hybridization and pulsed-field gel electrophoresis. A disruption mutant created by the insertion of a TRP1 marker into SRB1 displayed only the lysis ability phenotype and was not dependent on an osmotic stabilizer for growth. Lysis ability was acquired by growth in (or transfer to) an osmotically stabilized environment, but only under conditions which permitted budding. It is inferred that budding cells lyse with a higher probability and that weak points in the wall at the site of budding are involved in the process. The biotechnological potential of the cloned gene and the disruption mutant is discussed.
TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae.
Authors: Ko C H , Gaber R F
Published: October 2015

We describe the cloning and molecular analysis of TRK2, the gene likely to encode the low-affinity K+ transporter in Saccharomyces cerevisiae. TRK2 encodes a protein of 889 amino acids containing 12 putative membrane-spanning domains (M1 through M12), with a large hydrophilic region between M3 and M4. These structural features closely resemble those contained in TRK1, the high-affinity K+ transporter. TRK2 shares 55% amino acid sequence identity with TRK1. The putative membrane-spanning domains of TRK1 and TRK2 share the highest sequence conservation, while the large hydrophilic regions between M3 and M4 exhibit the greatest divergence. The different affinities of TRK1 trk2 delta cells and trk1 delta TRK2 cells for K+ underscore the functional independence of the high- and low-affinity transporters. TRK2 is nonessential in TRK1 or trk1 delta haploid cells. The viability of cells containing null mutations in both TRK1 and TRK2 reveals the existence of an additional, functionally independent potassium transporter(s). Cells deleted for both TRK1 and TRK2 are hypersensitive to low pH; they are severely limited in their ability to take up K+, particularly when faced with a large inward-facing H+ gradient, indicating that the K+ transporter(s) that remains in trk1 delta trk2 delta cells functions differently than those of the TRK class.
Derepression of mouse beta-major-globin gene transcription during erythroid differentiation.
Authors: Macleod K , Plumb M
Published: October 2015

Functional analysis of the mouse beta-major-globin gene promoter has revealed a negative regulatory element (-100 to -250 bp) which represses promoter activity in mouse erythroleukemia (MEL) cells. Promoter activity is induced 14-fold during terminal differentiation of MEL cells. Three major in vitro binding sites for NF1 (-250 bp), GATA-1 (-212 bp), and a sequence at -165 bp (BB1) have been defined in this region. Site-directed mutagenesis of any one of the three sites resulted in a five- to sixfold up-regulation of promoter activity in uninduced MEL cells, but only three- to fourfold stimulation was observed from the mutant promoters during MEL cell terminal differentiation. This finding suggests that all three sites are required for repressor activity in uninduced MEL cells and that derepression occurs during MEL cell differentiation. BB1 DNA-binding activity decreases during MEL cell differentiation, suggesting a central role for this factor in modulating the effects of the repressor element. The BB1-binding factor also competes with the CCAAT-binding protein for binding the CCAAT motif. The fact that a reduced but significant stimulation of promoter activity during differentiation is observed in the absence of the repressor element raises the possibility that the BB1 factor also down-regulates transcription in undifferentiated MEL cells by displacing binding of CCAAT-binding protein to the proximal CCAAT motif.
Leukemia inhibitory factor and interleukin-6 trigger the same immediate early response, including tyrosine phosphorylation, upon induction of myeloid leukemia differentiation.
Authors: Lord K A , Abdollahi A , Thomas S M , DeMarco M , Brugge J S , Hoffman-Liebermann B , Liebermann D A
Published: October 2015

Leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), two multifunctional cytokines lacking structural homology and binding to distinct receptors, share interesting functional similarities, which include induction of hematopoietic differentiation in normal and myeloid leukemia cells, induction of neuronal cell differentiation, and stimulation of acute-phase protein synthesis in hepatocytes. Structural information on the LIF receptor is not yet available, whereas recent cloning of the IL-6 receptor has shown it to be bipartite, with a signal-transducing subunit that lacks sequence homology to known protein kinases and produces second messengers of unknown nature. The molecular nature of the mechanisms which LIF and IL-6 use to induce cell differentiation is not known. To address this issue, we took advantage of a clone of M1 myeloblastic leukemia cells capable of being induced for terminal differentiation by both LIF and IL-6 directly activate the same set of immediate early response genes upon induction of M1 myeloid differentiation. At least two mechanisms of gene activation, one transcriptional and the other posttranscriptional, are shown to be involved. It is also shown that the LIF and IL-6 immediate early response, at suboptimal cytokine concentrations, is additive. Using a variety of protein kinase activators and inhibitors, we have shown that the intracellular signalling pathways for both LIF and IL-6 are distinct from those of known second messengers and involve protein phosphorylation, notably tyrosine phosphorylation of a 160-kDa protein, as an essential step(s) in the immediate early activation of MyD gene expression. These observations indicate that the functional similarities of LIF and IL-6 as inducers of cell differentiation prevail at the level of the complex differentiation immediate early response and implicate common mechanisms of signal transduction for LIF- and IL-6-induced differentiation.
Double-strand gap repair in a mammalian gene targeting reaction.
Authors: Valancius V , Smithies O
Published: October 2015

To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments.
Transactivation of pancreas-specific gene sequences in somatic cell hybrids.
Authors: Wu K J , Samuelson L C , Howard G , Meisler M H , Darlington G J
Published: October 2015

Enhancer/promoter elements from two pancreas-specific genes, those encoding amylase and elastase, were ligated to the bacterial GPT gene. The resulting construct can be used to select for expression of gene products which activate these pancreas-specific promoters in hybrid cells. The selectable GPT construct was stably transferred into several cell lines either directly or by cotransfection with pSV2Neo. GPT was expressed when transferred to pancreatic cell lines but not when transferred to GPT-fibroblast (L) cells or hepatoma cells. When the transformed L cells and hepatoma cells were fused with pancreatic cell lines, GPT was activated in the hybrid cells. Endogenous pancreas-specific genes from the L-cell and hepatoma parents were also activated in the hybrids. In addition, a pancreas-specific nuclear protein, PTF1, was produced in pancreatic and hybrid cells, correlating with GPT expression. The transformed L cells and hepatoma cells thus contained a nonexpressed construct which could be activated in trans by factors present in pancreatic cells. The hepatoma hybrid also continued to produce albumin, demonstrating the coexpression of liver and pancreas-specific genes in the hybrid-cell population. Cell lines carrying the amylase/elastase/GPT construct may be useful as a selection system for cloning of pancreatic transcription activators.
Differential ability of proximal and remote element pairs to cooperate in activating RNA polymerase II transcription.
Authors: Wang W D , Gralla J D
Published: October 2015

To investigate the synergism or cooperative interaction between transcription elements, we have designed and constructed a series of synthetic polymerase II promoters with different combinations of elements. These include three different CCAAT boxes, which correspond to the binding sites for CP1, CP2, and NFI, a GC box, a CACCC box, and an ATF/CREB-binding site. The synthetic promoters containing these elements in proximal positions were linked to a test gene (CAT). Tandem repeats of AP1- and AP2-binding sites, the simian virus 40 enhancer, and DNA-binding sites for GAL-estrogen receptor were cloned downstream of the test gene. The strength of these promoters was then tested in transient-expression assays in HeLa TK- cells. In the context of the adenovirus major late promoter TATA box, the promoters containing only certain combinations of elements are active in this assay. Some elements appear to cooperate nearly universally, but others exhibit strong selectivity. These results indicate strongly selective synergistic interactions between elements and suggest that levels of promoter strength may be determined by the extent of compatibility between factors bound to proximal and enhancer sites.
Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1.
Authors: Mitts M R , Bradshaw-Rouse J , Heideman W
Published: October 2015

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains many proteins, including the CYR1 polypeptide, which is responsible for catalyzing the formation of cyclic AMP from ATP, RAS1 and RAS2 polypeptides, which mediate stimulation of cyclic AMP synthesis by guanine nucleotides, and the yeast GTPase-activating protein analog IRA1. We have previously reported that adenylate cyclase is only peripherally bound to the yeast membrane. We have concluded that IRA1 is a strong candidate for a protein involved in anchoring adenylate cyclase to the membrane. We base this conclusion on the following criteria: (i) a disruption of the IRA1 gene produced a mutant with very low membrane-associated levels of adenylate cyclase activity, (ii) membranes made from these mutants were incapable of binding adenylate cyclase in vitro, (iii) IRA1 antibodies inhibit binding of adenylate cyclase to the membrane, and (iv) IRA1 and adenylate cyclase comigrate on Sepharose 4B.
Antifungal properties of the immunosuppressant FK-506: identification of an FK-506-responsive yeast gene distinct from FKB1.
Authors: Brizuela L , Chrebet G , Bostian K A , Parent S A
Published: October 2015

FK-506 is a novel and potent antagonist of T-cell activation and an inhibitor of fungal growth. Its immunosuppressive activity can be antagonized by the structurally related antibiotic rapamycin, and both compounds interact with cytoplasmic FK-506-binding proteins (FKBPs) in T cells and yeast cells. In this paper, we show that FK-506 and two analogs inhibit vegetative growth of Saccharomyces cerevisiae in a fashion that parallels the immunosuppressive activity of these compounds. Yeast mutants resistant to FK-506 were isolated, and at least three complementation groups (fkr1, fkr2, and fkr3) were defined. These fkr mutants show no alteration in their levels of FK-506-binding activity. Likewise, strains carrying null alleles of FKB1 (the yeast gene coding for the FKBP) remain FK-506 sensitive, indicating that depletion of yeast FKBP is not sufficient to confer an FK-506 resistance phenotype, although fkb1 null mutants are resistant to rapamycin. FKB1 does not map to the three fkr loci defined here. These results suggest that yeast FKBP mediates the inhibitory effect of rapamycin but that at least one other protein is directly involved in mediating the activity of FK-506. Interestingly, the ability of FK-506 to rescue a temperature-sensitive growth defect of the fkr3 mutant suggests that the FKR3 gene may define such a protein.
The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain.
Authors: Johnson D E , Lu J , Chen H , Werner S , Williams L T
Published: October 2015

To determine the mechanisms by which multiple forms of fibroblast growth factor (FGF) receptors are generated, we have mapped the arrangement of exons and introns in the human FGF receptor 1 (FGFR 1) gene (flg). We found three alternative exons encoding a portion of the third immunoglobulin (Ig)-like domain of the receptor. One of these alternatives encodes a sequence that is part of a secreted form of FGFR 1. The other two encode sequences that are likely part of transmembrane forms of FGFR 1. One of these forms has not been previously reported in published cDNAs. Also, we have determined the structural organization of a portion of the human FGFR 2 gene (bek) and found a similar arrangement of alternative exons for the third Ig-like domain. The arrangement of these genes suggests that there are conserved mechanisms governing the expression of secreted FGF receptors as well as the expression of at least two distinct membrane-spanning forms of the FGF receptors. The diverse forms appear to be generated by alternative splicing of mRNA and selective use of polyadenylation signals.
Tyrosine 706 and 807 phosphorylation site mutants in the murine colony-stimulating factor-1 receptor are unaffected in their ability to bind or phosphorylate phosphatidylinositol-3 kinase but show differential defects in their ability to induce early response gene transcription.
Authors: van der Geer P , Hunter T
Published: October 2015

The receptor for colony-stimulating factor-1 (CSF-1) is a receptor protein-tyrosine kinase. To study the possible function of CSF-1 receptor autophosphorylation, two autophosphorylation sites, Tyr-706, located in the kinase insert, and Tyr-807, a residue conserved in all protein-tyrosine kinases, were changed independently to either phenylalanine or glycine. Wild-type and mutant receptors were stably expressed in Rat-2 cells. In response to CSF-1, cells expressing Phe- or Gly-706 mutant receptors showed increased growth rate and altered cell morphology. Both the Phe- and Gly-706 mutant receptors associated with and phosphorylated phosphatidylinositol-3 kinase at levels comparable with those of wild-type receptors. However, these mutant receptors differed subtly from each other and from the wild-type receptor in their ability to induce different aspects of the response to CSF-1. The Phe-706 mutant receptor was most strongly affected in its ability to increase growth rate or elevate the levels of c-fos and NGF1A mRNAs, whereas the Gly-706 mutant receptor was most markedly affected in its ability to induce a change in cell morphology or increase the levels of c-jun and NGF1A mRNAs. These findings indicate that Tyr-706 itself, or this region of the receptor, may be important for interaction of the CSF-1 receptor with different signalling pathways. Gly-807 mutant receptors lacked protein-tyrosine kinase activity, failed to respond to CSF-1, and were defective in biosynthetic processing. Phe-807 mutant receptors had 40 to 60% reduced protein-tyrosine kinase activity in vitro. Although cells expressing Phe-807 receptors were able to respond to CSF-1, the changes in growth rate and cell morphology were significantly less than seen with wild-type receptors, and the induction of early response genes was also slightly lower than for the wild-type receptor. In contrast, Phe-807 receptors were equivalent to wild-type receptors when tested for their ability to interact with phosphatidylinositol-3 kinase. These findings indicate that phosphorylation of Tyr-807 may be important for full activation of the receptor.