bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel phytochrome sequences in Arabidopsis thaliana: structure, evolution, and differential expression of a plant regulatory photoreceptor family.
Authors: Sharrock R A , Quail P H
Published: June 2007

Phytochrome is a plant regulatory photoreceptor that mediates red light effects on a wide variety of physiological and molecular responses. DNA blot analysis indicates that the Arabidopsis thaliana genome contains four to five phytochrome-related gene sequences. We have isolated and sequenced cDNA clones corresponding to three of these genes and have deduced the amino acid sequence of the full-length polypeptide encoded in each case. One of these proteins (phyA) shows 65-80% amino acid sequence identity with the major, etiolated-tissue phytochrome apoproteins described previously in other plant species. The other two polypeptides (phyB and phyC) are unique in that they have low sequence identity (approximately 50%) with each other, with phyA, and with all previously described phytochromes. The phyA, phyB, and phyC proteins are of similar molecular mass, have related hydropathic profiles, and contain a conserved chromophore attachment region. However, the sequence comparison data indicate that the three phy genes diverged early in plant evolution, well before the divergence of the two major groups of angiosperms, the monocots and dicots. The steady-state level of the phyA transcript is high in dark-grown A. thaliana seedlings and is down-regulated by light. In contrast, the phyB and phyC transcripts are present at lower levels and are not strongly light-regulated. These findings indicate that the red/far light-responsive phytochrome photoreceptor system in A. thaliana, and perhaps in all higher plants, consists of a family of chromoproteins that are heterogeneous in structure and regulation.
NBP, a protein that specifically binds an enhancer of immunoglobulin gene rearrangement: purification and characterization.
Authors: Li M , Morzycka-Wroblewska E , Desiderio S V
Published: June 2007

Immunoglobulin and T-cell receptor (TCR) genes are encoded in discrete germ line DNA segments that are joined by site-specific recombination during lymphocyte development. These DNA rearrangements are mediated by conserved heptamer and nonamer DNA sequence elements that lie near the sites of recombination. In this paper we show that the nonamer element coincides with the recognition site for a specific DNA-binding protein: mutations within the nonamer sequence, but not outside of it, decrease affinity for the binding protein by 300- to 1000-fold. Deletion of the binding site for the protein results in at least a 50-fold decrease in recombination frequency in vivo. By a combination of conventional and recognition site affinity chromatography, we have achieved greater than 20,000-fold purification of the protein from calf thymus, with an overall yield of 22%. The purified protein, which we now call nonamer-binding protein (NBP), has an apparent molecular weight of 63,000 and a frictional ratio of 1.27, suggesting that it exists as a globular monomer in 0.5 M NaCl. Our observations suggest that NBP is a component of the recombinational apparatus.
The promoter-proximal rDNA terminator augments initiation by preventing disruption of the stable transcription complex caused by polymerase read-in.
Authors: Henderson S L , Ryan K , Sollner-Webb B
Published: June 2007

We have examined the mechanism by which transcriptional initiation at the mouse rDNA promoter is augmented by the RNA polymerase I terminator element that resides just upstream of it. Using templates in which terminator elements are instead positioned at the opposite side of the plasmid rather than proximal to the promoter, or conditions where transcription is terminated elsewhere in the plasmid by UV-induced lesions, we show that the terminator's stimulatory effect is not position dependent. Mouse terminator elements therefore do not stimulate via the previously postulated 'read-through enhancement' model in which terminated polymerases are handed off to an adjacent promoter in a concerted reaction. The position independence and orientation dependence of the terminator also makes it unlikely that the terminator functions as a promoter element or as an enhancer. Instead, terminators serve to augment initiation by preventing polymerases from reading completely around the plasmid and through the promoter from upstream, an event which we show interferes with subsequent rounds of initiation. Notably, this transcriptional interference arises because polymerase passage across a promoter disrupts the otherwise stable transcription complex, specifically releasing the bound transcription factor D. These liberated D molecules can then bind to other templates and activate their expression. The rDNA transcriptional interference is not due to a steric impediment to the binding of new polymerase molecules, and it does not similarly liberate the initiation-competent polymerase (factor C). These studies have also convincingly demonstrated that multiple rounds of transcription are obtained from rDNA template molecules in vitro.
Structure and expression of a family of Ultrabithorax mRNAs generated by alternative splicing and polyadenylation in Drosophila.
Authors: Kornfeld K , Saint R B , Beachy P A , Harte P J , Peattie D A , Hogness D S
Published: June 2007

The 77-kb primary transcript of the homeotic Ultrabithorax (Ubx) gene is alternatively spliced to yield at least five different coding regions. Each is restricted to either a 3.2- or a 4.3-kb size class generated by alternative polyadenylation. The pathways for splicing and polyadenylation are therefore coordinately regulated, and because the relative abundance of the respective mRNAs varies throughout development, these pathways also appear to be developmentally regulated. Translation of these mRNAs yields a family of Ubx proteins characterized by constant amino- and carboxy-proximal regions of 247 and 99 amino acid residues, respectively. Members of this family are distinguished by a short variable region that links the constant regions and consists of different combinations of three optional elements of 9, 17, and 17 residues. Only four amino acid residues separate this variable region from the 60-residue homeo domain of the carboxy-terminal constant region. This proximity suggests that functional differences among the Ubx proteins derive from the differential effects of their variable regions on the DNA-binding capacity of the homeo domain. An argument is made that these functional differences are tissue specific.
In vitro formation of short RNA polymerase II transcripts that terminate within the HIV-1 and HIV-2 promoter-proximal downstream regions.
Authors: Toohey M G , Jones K A
Published: June 2007

Trans-activation of HIV-1 transcription by the viral regulatory protein Tat has been proposed recently to overcome a block to RNA polymerase II elongation in vivo imposed by 5'-untranslated leader sequences. Interestingly, however, only full-length transcripts, rather than prematurely terminated HIV RNAs, are synthesized in most cell-free transcription extracts. Here, we describe an in vitro system in which induction of a highly efficient RNA polymerase II termination or cotranscriptional RNA processing event creates short HIV RNAs with 3' ends that map to a region immediately downstream of the HIV-1 or HIV-2 promoters. Termination in vitro is sequence dependent, generating short HIV-1 RNAs of 58-61 nucleotides that resemble in vivo transcripts observed in the absence of Tat, and a distinct, longer transcript of approximately 125-130 nucleotides from the HIV-2 promoter. Deletion of promoter-proximal HIV-2 downstream sequences results in the loss of a discrete RNA but also fails to restore wild-type transcription, indicating that termination actually is specified at the promoter and occurs at a site positioned by one or more elements located immediately upstream of the 3' end of the short RNAs. Experiments with recombinant HIV-2 promoters and nucleoside analogs indicate that this event involves a concerted interaction between the promoter and orientation-dependent leader sequences and that RNA secondary structure formation may also be required. These data provide direct evidence for abbreviated HIV transcripts and an in vitro approach to understanding the roles of cellular and viral regulatory proteins that mediate this process at the HIV promoters.
Yeast YAP1 encodes a novel form of the jun family of transcriptional activator proteins.
Authors: Moye-Rowley W S , Harshman K D , Parker C S
Published: June 2007

The jun family of transcriptional activators includes mammalian AP-1 as well as the yeast regulatory protein GCN4. Recently, an additional transcriptional activator has been found in yeast that recognizes the TGACTCA sequence element common in GCN4/AP-1 sites. This factor was designated yAP-1. The structural gene for yAP-1 has now been isolated and characterized. The deduced amino acid sequence predicts a protein of 650 residues, considerably larger than GCN4 or c-Jun. The amino terminus of yAP-1 is homologous to the carboxy-terminal DNA-binding domains of GCN4 and c-Jun. Disruption of the YAP1 gene demonstrates this gene is not essential but is required for AP-1 recognition element-dependent transcriptional activation. DNA-affinity blots of proteins from YAP1 cells suggest the presence of additional TGACTCA-binding proteins other than GCN4 and yAP-1. Furthermore, expression of at least one of these related DNA-binding proteins appears to be under control of yAP-1.
A FOS protein is present in a complex that binds a negative regulator of MYC.
Authors: Hay N , Takimoto M , Bishop J M
Published: June 2007

Regulation of the human proto-oncogene MYC apparently plays an important role in cellular proliferation and the genesis of diverse tumors. Transcription from MYC is governed principally by two promoters known as P1 and P2. Previously we have detected a negative regulator of these promoters upstream of MYC. We now report that this regulator comprises no more than 26 bp of DNA, with sequence that resembles the regulators of at least two other genes, and we describe nuclear factors that interact with the regulator. Nuclear extracts from human cells form three distinctive complexes with the negative regulator. One of these complexes includes the product of the proto-oncogene FOS or an antigenically related protein, and the FOS protein may, in turn, be associated with the product of the proto-oncogene JUN. Similarly, FOS and JUN proteins produced by translation in vitro bind cooperatively to the negative regulator. These results raise the possibility that FOS and JUN participate in the regulation of MYC.
Growth factors and membrane depolarization activate distinct programs of early response gene expression: dissociation of fos and jun induction.
Authors: Bartel D P , Sheng M , Lau L F , Greenberg M E
Published: June 2007

A set of early response genes has been identified whose transcription in fibroblasts is rapidly induced in response to growth factors. Prototype members of this group, c-fos and c-jun, encode products that form a heterodimer and have been implicated in the regulation of gene expression and cell growth. It is thought that other early response genes also encode critical mediators of the cell's response to external stimuli. We have used PC12 pheochromocytoma cells as a model system to test the hypothesis that different extracellular signals induce distinct patterns of expression of early response genes. Our results indicate that membrane depolarization, induced either by potassium chloride or by the neurotransmitter analog nicotine, activates a program of gene expression distinct from that activated by nerve growth factor or epidermal growth factor. Notably, c-fos and c-jun activation can be dissociated; whereas c-jun is coinduced with c-fos and jun-B after growth factor stimulation, membrane depolarization activates c-fos and jun-B without stimulating c-jun. Fos may therefore form transcription complexes with alternative cofactors under different stimulation conditions. nur/77 and zif/268, which encode putative transcription factors, also show markedly different responses to growth factors and depolarization. We conclude that multiple nonconvergent signal transduction pathways control early response gene expression. Our findings also indicate that the diversity and specificity of cellular response to environmental change can be accounted for by the differential combinatorial induction of a relatively small number of early response genes.
Nucleolin from Xenopus laevis: cDNA cloning and expression during development.
Authors: Caizergues-Ferrer M , Mariottini P , Curie C , Lapeyre B , Gas N , Amalric F , Amaldi F
Published: June 2007

Nucleolin is a key nucleolar protein in higher eukaryotic cells and is involved directly in ribosome biogenesis. Using an antiserum raised against hamster nucleolin, the homologous protein was detected in nucleoli of Xenopus laevis hepatocytes as well as in the amplified nucleoli of oocytes. A cDNA encoding Xenopus nucleolin has been isolated and sequenced. The deduced protein sequence reveals similar domains in Xenopus and in mammals, but they have undergone separate evolutions. In particular, each of the four RNA-binding domains has evolved differently--the carboxy-proximal domain is twice as conserved (87%) as the amino-proximal domain (42%). These data shed some light on the possible roles of each domain. The expression of nucleolin has been followed throughout oogenesis and embryogenesis. The appearance of nucleolin during early development precedes the transcription of rDNA and the synthesis of ribosomal proteins. The maximal accumulation of nucleolin at gastrulation coincides with nucleolar reformation. Furthermore, when ribosomal synthesis is activated during oogenesis and embryogenesis, peptides immunorelated to nucleolin appear and accumulate. The results suggest that nucleolin plays a role not only in ribosome assembly but also in nucleologenesis.
Isolation, structure, and expression of labial, a homeotic gene of the Antennapedia Complex involved in Drosophila head development.
Authors: Diederich R J , Merrill V K , Pultz M A , Kaufman T C
Published: June 2007

The labial (lab) gene of Drosophila melanogaster is necessary for the proper development of the embryonic (larval) and adult head. We have identified the lab transcription unit within the proximal portion of the Antennapedia Complex (ANT-C) by mapping the molecular lesions associated with chromosomally rearranged lab alleles. We present its molecular structure, nucleotide sequence, and temporal pattern of expression. In addition, using antibodies generated against a fusion protein, we show that in the embryo the lab protein is distributed in neural and epidermal cells of the procephalic lobe; in a discrete loop of the midgut; and in specific progenitor sensory cells of the clypeolabrum, thoracic segments, and tail region. The regions of lab expression in the developing cephalon represent nonsegmented domains that are anterior to and largely nonoverlapping with the domains of expression of the Deformed (Dfd) and proboscipedia (pb) genes, two other homeotic loci of the ANT-C that also function to direct the development of head structures. Furthermore, lab head expression is associated with the complex cellular movements of head involution, a process that not only is defective in lab embryos, but the failure of which appears to be largely responsible for the defects observed in mutant embryos. Finally, we suggest that lab head expression provides a molecular marker for an intercalary segment, an ancestral segment that has become morphologically indistinct during the evolution of the insect head.
Unusual transcriptional and translational features of the aminoglycoside phosphotransferase gene (aph) from Streptomyces fradiae.
Authors: Janssen G R , Ward J M , Bibb M J
Published: June 2007

The aminoglycoside phosphotransferase gene (aph) from the neomycin producer Streptomyces fradiae encodes an enzyme (APH) that phosphorylates, and thereby inactivates, the antibiotic neomycin. Two promoters were identified upstream of and oriented toward the aph coding sequence. One promoter (aphp1) initiated transcription at the A of the ATG translational initiation codon, or one to two bases upstream. Mutations made in this promoter region identified functionally important nucleotides and verified that the aphp1 transcript was translated to yield the APH protein, despite the lack of a conventional ribosome binding site. A second aph promoter, aphp2, initiated transcription 315 bp upstream of the translational initiation codon but gave transcripts that appeared to terminate before reaching the coding sequence. Multiple transcriptional initiation sites (pA1-pA5) were identified also in the aph regulatory region oriented in the opposite direction to aph transcription. Promoters for the pA2 and pA4 transcripts overlap with aphp1 such that down-promoter mutations in aphp1 also reduce transcription from the overlapping pA promoters.
Alteration of hsp82 gene expression by the gypsy transposon and suppressor genes in Drosophila melanogaster.
Authors: Dorsett D , Viglianti G A , Rutledge B J , Meselson M
Published: June 2007

Several mutations in Drosophila result from insertion of the gypsy retrotransposon. Gypsy insertion mutagenesis and its modulation by allele-specific modifier genes were investigated by inserting gypsy or fragments of it into the intron of the Drosophila hsp82 heat shock gene. With gypsy in the parallel orientation, nearly all transcripts in transfected cells and transformed pupae were truncated in the 5' long terminal repeat (LTR). Truncation also occurred in or near the 3' LTR. The 5' LTR polyadenylation signal was strongly potentiated by a downstream 326-bp internal gypsy segment in either orientation. Anti-parallel gypsy reduced the amount of normal transcript to a much smaller extent, and a low level of truncation occurred within gypsy. No evidence was found for effects of the gypsy insertions on the hsp82 promoter. Mutations in the allelespecific modifier genes su(f) and su(w alpha) had effects on the amounts of readthrough transcripts consistent with their genetic behavior, whereas the effects of mutations in su(Hw) were only partly in accord with genetic expectations.
In vitro reconstitution of snRNPs: a reconstituted U4/U6 snRNP participates in splicing complex formation.
Authors: Pikielny C W , Bindereif A , Green M R
Published: June 2007

We have reconstituted in vitro the four snRNPs known to be involved in pre-mRNA splicing: U1, U2, U5, and U4/6. Reconstitution involves adding either authentic or in vitro-synthesized snRNAs to extracts enriched in snRNP structural polypeptides. The reconstituted snRNPs have the same buoyant density and are immunoprecipitated by the same antibodies as authentic snRNPs. Thus, the polypeptide composition of reconstituted snRNPs is similar, if not identical, to that of authentic snRNPs. We show further that a reconstituted U4/U6 particle is fully functional in forming splicing complexes with pre-mRNA. As is the case for the authentic U4/U6 snRNP, the reconstituted U4 snRNP, but not the U6 snRNA, dissociates from the complex prior to formation of the mature spliceosome. The ability to reconstitute snRNPs and assay their activity in spliceosome formation should provide a powerful approach to study these particles.
The organization and expression of a maize ribosomal protein gene family.
Authors: Larkin J C , Hunsperger J P , Culley D , Rubenstein I , Silflow C D
Published: June 2007

We have isolated several Zea mays cDNAs encoding the 40S subunit ribosomal protein S14. In maize, this ribosomal protein is encoded by a small multigene family, at least three members of which are expressed. S14 transcript levels are highest in mitotically active tissues, such as seedling shoot, developing endosperm, and tassel primordia, and lowest in tissues with little cell division, such as mature leaf and root. Very little S14 RNA is present in pollen, suggesting that translation of pollen mRNAs during pollen germination uses preformed ribosomes. During kernel development, the highest levels of S14 transcripts in endosperm tissue are found at 10-12 days postpollination; S14 RNA levels decline continuously from this point onward. The period of maximal expression of the S14 ribosomal protein gene appears to precede the onset of storage protein synthesis and does not correlate with the reported times of increased nucleolar volume or genome amplification.
Specific recognition nucleotides and their DNA context determine the affinity of E2 protein for 17 binding sites in the BPV-1 genome.
Authors: Li R , Knight J , Bream G , Stenlund A , Botchan M
Published: June 2007

The DNA context of nucleotides that a protein recognizes can influence the strength of the protein-DNA interaction. Moreover, in prokaryotes, understanding the quantitative differences in binding affinities that result in part from the DNA context is often important in describing regulatory mechanisms. Nevertheless, these issues have not been a major focus yet for the investigation of protein-DNA interactions in eukaryotes. In this study, we explored the binding specificity and the range of affinities that the BPV-1 E2 transcriptional activator has for DNA. Because E2 binding sites are positioned near several different BPV-1 promoters, such quantitative information may be important to understand transcriptional regulatory mechanisms in BPV-1. Gel retardation assays and DNA footprinting were used to quantitate the affinities of the E2 binding sites in the viral genome. In the process, five sites were discovered, which, on the basis of sequence, had not been predicted previously to interact with the E2 protein. Equilibrium and kinetic studies show that the range of E2 affinities of the 17 sites varied over 300-fold. The sequence elements responsible for E2 recognition of DNA were determined by missing contact analysis of several sites and a point mutation analysis of one site. The results presented show that the affinity of an E2 binding site is to a large extent determined by the availability of specific contacts, but the data also strongly suggest that DNA structure plays an important role.
CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation.
Authors: Iguchi-Ariga S M , Schaffner W
Published: June 2007

In mammals and other vertebrates, cytosine methylation in CpG sites is often negatively correlated with gene activity. Because methylation of the promoter region is most crucial for this effect, the simplest hypothesis is that CpG methylation interferes with the binding of specific transcription factors. We have examined this hypothesis with two different transcription factor-binding sites that contain a CpG dinucleotide, namely the cAMP-responsive element (CRE; 5'-TGACGTCA) and the Sp1-binding site (5'-GTGAGGCGGTGAGACT). We have reported previously that CpG methylation of the Sp1-binding site affected neither factor binding nor transcription in HeLa cells, which may be related to the fact that Sp1 is typically associated with promoters of housekeeping genes. In contrast, CREs are often associated with promoters of cell type-specific genes. A synthetic oligonucleotide containing two tandem CREs derived from the gene encoding the human glycoprotein hormone alpha-subunit was cloned upstream of a reporter gene. Transcription of this gene was dependent on the CRE sequences in both PC12 and HeLa cells. Bandshift and methylation interference assays show that similar, if not the same, factor(s) bind to the CRE in both cell lines, even though induction by cAMP was only observed in PC12 cells. CpG methylation of the CRE consensus sequences (TGACGTCA) resulted in loss of specific factor binding, as well as loss of transcriptional activity in vitro and in vivo, in both cell types. This suggests that the inactivity of methylated promoters can, at least in some cases, be explained by their inability to bind specific transcription factors.
Complementary homeo protein gradients in developing limb buds.
Authors: Oliver G , Sidell N , Fiske W , Heinzmann C , Mohandas T , Sparkes R S , De Robertis E M
Published: June 2007

A new human homeo box-containing gene designated Hox-5.2 was cloned and mapped to human chromosome 2. This homeo box is related in sequence to Abdominal-B, a Drosophila homeotic gene that specifies identity of posterior segments. An antibody probe was made using a human Hox-5.2 fusion protein and was found to stain posterior regions of mouse, chicken, and Xenopus embryos. Unexpectedly, when the distribution of Hox-5.2 antigen was compared with that of X1Hbox 1 antigen, a non-overlapping and mutually exclusive pattern was detected (e.g., in developing limb buds, intestine, and somites). Regions expressing Hox-5.2 do not express X1Hbox 1 protein, and vice versa. Hox-5.2 antigen is detected strongly in developing fore- and hindlimb buds, where it forms a gradient of nuclear protein throughout most of the mesenchyme. This gradient is maximal in distal and posterior regions. Hox-5.2 expression is activated in Xenopus limb regeneration blastemas, as expected for any gene involved in pattern formation. As described previously, a gradient of X1Hbox 1 protein can be detected in the forelimb. The latter gradient has the opposite polarity to that of Hox-5.2. i.e., maximal in anterior and proximal mesoderm. These two opposing gradients (and possibly others) could be involved in determining positional values in developing limb buds.
Analysis of the cis-acting requirements for germ-line-specific splicing of the P-element ORF2-ORF3 intron.
Authors: Laski F A , Rubin G M
Published: June 2007

P-element transposition is limited to the germ line because the element's third intron is only spliced in germ line cells. We show that a 240-bp fragment containing this 190-bp intron can confer germ line specificity when placed in the context of another gene. We find that the cis-acting regulatory sequences required for germ line regulation map near to, but not at, the 5' or 3' splice junctions.
cis-acting DNA sequence requirements for P-element transposition.
Authors: Mullins M C , Rio D C , Rubin G M
Published: June 2007

The P transposable element of Drosophila melanogaster has a complex array of cis-acting DNA sequences necessary for efficient transposition. At the 3' end these sequences extend over more than 150 bp and include 11- and 31-bp sequences found repeated in inverted orientation at the 5' end. The P element's 5' end, however, cannot function as its 3' end. When two 3' P-element ends are present, the more proximal end is used preferentially. We found also that the duplication of the target site does not appear to play a role in forward transposition.
The Drosophila E74 promoter contains essential sequences downstream from the start site of transcription.
Authors: Thummel C S
Published: June 2007

The Drosophila E74 gene is one of a small set of genes induced directly by the steroid hormone ecdysone at the onset of metamorphosis. Both in vitro and in vivo transcription assays have been used to delineate the promoter for the 6-kb E74 mRNA. Sequences upstream from position -83 had little effect on the amount of RNA synthesized in vitro, using extracts prepared from Drosophila Kc tissue culture cells. Deletion of a 5'-flanking TATA consensus sequence had no effect on the accuracy of transcriptional initiation and resulted in an increase in RNA synthesis, suggesting removal of a repressor binding site. Surprisingly, removal of the first two nucleotides of the transcribed region still allowed relatively high levels of transcription from the correct start site position. Removal of five additional nucleotides inactivated the promoter. In vitro transcription of a series of 3' deletions defined the 3' in vitro promoter boundary at position +43. Additional 5'-flanking sequences, between -181 and -83, were found to be necessary for efficient transcription in transfected Kc tissue culture cells. Two transcription factors that interact with the E74 promoter, zeste and GAGA, were studied in DNA-binding assays. zeste binds to two sites within the E74 promoter. These sites overlap with three of the six GAGA-binding sites. The zeste- and GAGA-binding sites lie within domains identified by deletion mapping as cis-acting transcriptional control elements.
Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element.
Authors: McGrew L L , Dworkin-Rastl E , Dworkin M B , Richter J D
Published: June 2007

Xenopus oocytes contain several mRNAs that are mobilized into polysomes only at the completion of meiosis (maturation) or at specific times following fertilization. To investigate the mechanisms that control translation during early development, we have focused on an mRNA, termed G10, that is recruited for translation during oocyte maturation. Coincident with its translation, the poly(A) tail of this message is elongated from approximately 90 to 200 adenylate residues. To identify the cis sequence that is required for this cytoplasmic adenylation and recruitment, we have synthesized wild-type and deletion mutant G10 mRNAs with SP6 polymerase. When injected into oocytes that subsequently were induced to mature with progesterone, wild-type G10 mRNA, but not mutant transcripts lacking a 50-base sequence in the 3'-untranslated region, was polyadenylated and recruited for translation. The 50-base sequence was sufficient to confer polyadenylation and translation when fused to globin mRNA, which does not normally undergo these processes during oocyte maturation. Further mutational analysis of this region revealed that a U-rich sequence 5' to the AAUAAA hexanucleotide nuclear polyadenylation signal, as well as the hexanucleotide itself, were both required for polyadenylation and translation. The 50-base cis element directs polyadenylation, but not translation per se, as a transcript that terminates with 3'-deoxyadenosine (cordycepin) is not recruited for translation. The available data suggest that the dynamic process of polyadenylation, and not the length of the poly(A) tail, is required for translational recruitment during oocyte maturation.
Expression of v-src induces a myeloproliferative disease in bone-marrow-reconstituted mice.
Authors: Keller G , Wagner E F
Published: June 2007

A recombinant retrovirus, N-TK-src, was used to introduce the v-src oncogene into mouse hematopoietic cells. This vector efficiently expresses both the neo and v-src genes in different hematopoietic lineages in culture as well as in mice reconstituted with infected bone marrow cells. Expression of v-src had no dramatic effect on the proliferative and differentiative capacity of hematopoietic precursors when assayed in methyl cellulose cultures. However, in mice reconstituted with N-TK-src-infected bone marrow cells, expression of v-src leads to the rapid development of a severe myeloproliferative disease, characterized by splenomegaly, anemia, and a shift of hematopoiesis from the bone marrow to the spleen.
Selection for B cells with productive IgL gene rearrangements occurs in the bursa of Fabricius during chicken embryonic development.
Authors: McCormack W T , Tjoelker L W , Barth C F , Carlson L M , Petryniak B , Humphries E H , Thompson C B
Published: June 2007

The vast majority of immunoglobulin-expressing mature chicken B lymphocytes contain one functionally rearranged and one unrearranged allele of the immunoglobulin light chain (IgL) gene. Therefore, nearly all IgL V-J rearrangements present in mature chickens are in-frame. In contrast, the Ig genes of mature mammalian B cells contain a high proportion of out-of-frame V-J joints. To investigate the basis for this difference, gene rearrangement at the chicken IgL locus was characterized during embryonic development and in mature B-cell lines. Joining of the single functional variable (VL) segment with the single joining (JL) segment occurs in cells in multiple tissues during a transient period of chicken embryogenesis. Only one-third of the V-J joints cloned from days 10-12 of development are in-frame. An increasing proportion of in-frame V-J joints is observed within the bursa of Fabricius at successively later stages of development. Our data suggest that the bursa of Fabricius serves during embryonic development as a site of selective amplification of cells that have undergone productive V-J joining, such that nearly all V-J joints present in postembryonic B cells are in-frame. The high frequency of rearranged alleles joined in-frame that is found in posthatching bursal cells and mature B-cell lines appears to result from a low frequency with which cells undergo IgL rearrangement at both alleles, rather than from an increase in the precision of V-J joining in avian species.
mec-7 is a beta-tubulin gene required for the production of 15-protofilament microtubules in Caenorhabditis elegans.
Authors: Savage C , Hamelin M , Culotti J G , Coulson A , Albertson D G , Chalfie M
Published: June 2007

In the nematode Caenorhabditis elegans, microtubules with 15 protofilaments are a specialized feature of six touch-receptor neurons; microtubules found in other C. elegans neurons have 11 protofilaments. Mutations in the gene mec-7 result in touch-insensitive animals whose touch cells lack the 15-protofilament microtubules. We have characterized 54 mutations in the mec-7 gene. The absence of mec-7 activity results selectively in the recessive loss of touch sensitivity. Partial loss-of-function alleles result in a partial loss of touch sensitivity. Dominant mutations, which are isolated at an unusually high proportion, may encode abnormal products. We have cloned the mec-7 gene; it encodes a beta-tubulin which is 90-93% identical to vertebrate beta-tubulin. Our results are consistent with the hypothesis that tubulin heterogeneity contributes to the formation of structurally and functionally distinct sets of microtubules.
Regulated expression at high copy number allows production of a growth-inhibitory oncogene product in Drosophila Schneider cells.
Authors: Johansen H , van der Straten A , Sweet R , Otto E , Maroni G , Rosenberg M
Published: June 2007

The Drosophila metallothionein promoter (Mtn) was used to obtain efficient, regulated expression of foreign gene products inserted in high copy numbers into Drosophila melanogaster Schneider 2 cells. An expression unit comprised of a reporter gene [Escherichia coli galactokinase (galK)] fused to the Mtn promoter was stably introduced into Schneider 2 cells in up to several hundred copies per cell in a single transfection-selection event. This system contrasts dramatically with other eukaryotic systems that permit only a few copies of a gene to be stably inserted in a single transfection-selection event. The transfected Drosophila S2 cell lines expressed high levels of both galK mRNA and protein in response to metal induction. Most important, and in contrast to mammalian cells, expression remained fully regulated even at high copy number, with low basal expression maintained in the absence of inducer. This regulated system was used to obtain efficient expression in Drosophila cells of an otherwise lethal or growth-inhibitory gene product, the human H-ras oncogene. The ability to obtain regulated high-level expression of potentially lethal foreign proteins is unique to the Drosophila cell system.
Isolation and characterization of a gene specifically expressed in different metastatic cells and whose deduced gene product has a high degree of homology to a Ca2+-binding protein family.
Authors: Ebralidze A , Tulchinsky E , Grigorian M , Afanasyeva A , Senin V , Revazova E , Lukanidin E
Published: June 2007

The gene mts1, which is expressed specifically in metastatic cells, was isolated by molecular cloning coupled with differential DNA reassociation. Transcription of mts1 was found not only in tumor cells, but also in normal cells; homologous RNA was detected only in spleen, thymus, bone marrow, and blood lymphocytes. DNA sequencing of mts1 revealed an open reading frame containing information for a peptide of 101 amino acids, and the amino acid sequence suggested that the mts1 protein was identical to the previously isolated Ca2+-binding mouse protein (Jackson-Grusby et al. 1987; Goto et al. 1988). Thus, the mts1 protein is a member of the calcium-modulated protein family, and our data indicate that mts1 is involved in regulating the metastatic behavior of tumor cells.
A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes.
Authors: Passmore S , Elble R , Tye B K
Published: June 2007

The Saccharomyces cerevisiae MCM1 gene product is a protein with multiple functions. It is a transcription factor necessary for expression of mating-type-specific genes and is also required for the maintenance of minichromosomes. MCM1 shows DNA-binding specificities similar to those of two previously reported DNA-binding factors, pheromone/receptor transcription factor (PRTF) and general regulator of mating type (GRM); like PRTF, its activity can be modulated by the alpha 1 protein. MCM1 binds to the dyad symmetry element 5'-CCTAATTAGG and related sequences, which we refer to as MCM1 control elements (MCEs). MCEs are found within the regulatory regions of a- and alpha-specific genes. Direct and indirect DNA binding assays suggest that a conserved 5'-ATTAGG in one-half of the dyad symmetry element is important for MCM1 binding whereas variants in the other half are tolerated. We have used a novel DNase I 'nicking interference' assay to investigate the interaction of MCM1 with its substrate. These data suggest that MCM1 binds as a dimer, interacting symmetrically with the ATTAGG residues in each half of the binding site. MCM1 contains striking homology to the DNA-binding domain of the human serum response factor (SRF) which mediates the transient transcriptional activation of growth-stimulated genes by binding to the serum response element (SRE). We have shown that MCM1 binds to the human c-fos SRE in vitro and, like other MCEs, the c-fos SRE exhibits MCM1-mediated upstream activating sequence (UAS) activity in vivo.
The yeast transcription activator PRTF, a homolog of the mammalian serum response factor, is encoded by the MCM1 gene.
Authors: Jarvis E E , Clark K L , Sprague G F
Published: June 2007

Two proteins, alpha 1 and pheromone/receptor transcription factor (PRTF), bind cooperatively to the upstream activation sequences (UAS) of yeast alpha-specific genes and thereby activate their transcription. In these protein-DNA complexes, the PRTF moiety interacts with a degenerate dyad symmetric sequence, the P box. PRTF contributes also to the regulation of a second set of cell-type-specific genes, the a-specific genes. We used two in vitro assays to show that PRTF is encoded, at least in part, by the MCM1 gene. In one assay, truncated MCM1 proteins encoded by deletion derivatives of the MCM1 gene formed protein-DNA complexes of novel mobility, demonstrated that MCM1 can bind to the P-box-containing DNA. Second, antibodies raised to a synthetic MCM1 polypeptide retard the migration of PRTF-DNA complexes in gel mobility shift assays. This result indicates that PRTF, defined as an activity that binds cooperatively with alpha 1 to alpha-specific UAS elements, shares an epitope with MCM1. In addition, we show that MCM1 deletions that remove the carboxy-terminal 129 codons of 286 total codons encode truncated MCM1 molecules that are competent to activate transcription in vivo, indicating that the carboxy-terminal residues are not required for this process.
An avian muscle factor related to MyoD1 activates muscle-specific promoters in nonmuscle cells of different germ-layer origin and in BrdU-treated myoblasts.
Authors: Lin Z Y , Dechesne C A , Eldridge J , Paterson B M
Published: June 2007

We isolated the cDNA encoding a myogenic factor expressed in embryonic chick breast muscle by virtue of its weak hybridization to the mouse MyoD1 clone. Nucleotide sequence analysis and amino acid comparison define this clone, CMD1, as encoding a protein similar to mouse MyoD1. CMD1 encodes a polypeptide smaller than MyoD1, 298 versus 318 amino acids, respectively, and is 80% concordant by amino acid sequence overall. The basic and myc domains required for myogenic conversion of mouse 10T1/2 'fibroblasts' to myoblasts with MyoD1 are completely conserved in CMD1. CMD1 is just as efficient as the mouse homolog in myogenic conversion of 10T1/2 cells and coactivates the endogenous mouse MyoD1 gene in the process. The efficiency of myoblast conversion depends on the levels of CMD1 expression and suggests that the cellular concentration of CMD1 plays a role in the onset of myogenesis. Transient expression of CMD1 in a variety of nonmuscle cells from different germ-layer origins activates both cotransfected muscle-specific promoters and, in some cases, endogenous muscle-specific genes. 5-Bromodeoxyuridine (BrdU) treatment of chicken and mouse myoblasts reduces the expression of CMD1 and MyoD1, respectively, and may explain how this thymidine analog inhibits myogenesis and the activity of transfected muscle-specific promoters in BrdU-treated myoblasts. Transient expression of CMD1 in BrdU-treated myoblasts reactivates cotransfected muscle-specific promoters. CMD1 activates muscle-specific promoters in cotransfections regardless of cell type, whereas 'housekeeping' or constitutive promoters can be activated moderately, unaffected, or repressed, depending on the promoter and cell background. The rate and degree of myogenic conversion may be more restricted by cell phenotype than by germ-layer origin.
Structure and distribution of the Notch protein in developing Drosophila.
Authors: Kidd S , Baylies M K , Gasic G P , Young M W
Published: June 2007

Antibodies to Notch show that it is a stable, high-molecular-weight transmembrane glycoprotein, with epidermal growth factor (EGF)-like elements exposed on the cell surface. The protein is phosphorylated variably on serines of the cytoplasmic domain. Individual Notch polypeptide chains appear to be associated with one another by disulfide bonds, suggesting that homotypic interaction of these proteins is required for function. Immunocytochemistry has revealed striking features of Notch expression that might clarify its function: Cells of the ventral neurogenic ectoderm become conspicuously labeled with the protein prior to embryonic neurogenesis, and Notch appears to be associated with cells destined for both neural and epidermal lineages. High levels of Notch become restricted to neuroblasts as they delaminate from the embryonic ectoderm and are apposed to mesoderm. Mesodermal cells express Notch also, suggesting a possible involvement in neurogenesis, or an unknown role in mesoderm differentiation. In larvae and pupae, a correlation of expression and neuroblast mitotic activity is seen for many cells. Notch produced by a dividing neuroblast may persist on derivative cells, including terminally differentiated neurons and nerve processes. In the larval eye imaginal disk, strong Notch expression appears in the morphogenetic furrow, uniformly on cell surfaces as they cluster to form ommatidia. Expression persists on ommatidia after release from the furrow. These patterns suggest a role for Notch in position-dependent development in both initiation and maintenance of cell-surface interactions. In the eye and embryonic ectoderm, uniform expression on cells interacting to produce different developmental lineages from a single primordium suggests that Notch alone may not be sufficient to elaborate cell fates.
Embryonic expression pattern of a family of Drosophila proteins that interact with a central nervous system regulatory element.
Authors: Bray S J , Burke B , Brown N H , Hirsh J
Published: June 2007

The protein Elf-1 interacts with a cis-acting element that is required specifically for the neuronal expression of the Drosophila dopa decarboxylase gene Ddc. Using protein purified from Drosophila embryos, we raised Elf-1-specific monoclonal antibodies. The expression of Elf-1 during embryogenesis is restricted to nuclei of tissues derived from ectoderm, predominantly the central nervous system (CNS) and the epidermis. Within the CNS, Elf-1 is present in only a small fraction of nuclei, and the pattern of expressing nuclei changes dramatically during development. Elf-1 and Ddc are coexpressed in primary cultures of neural cells. However, we do not detect Elf-1 in Ddc-expressing neurons in vivo, leading to the suggestion that Elf-1 activity is required in vivo for initiation of Ddc expression but not for its maintenance. The antibodies also were used to isolate cDNA clones encoding Elf-1. Alternate forms of Elf-1 mRNA result in at least three protein isoforms.
Regulated phosphorylation and dephosphorylation of GAL4, a transcriptional activator.
Authors: Mylin L M , Bhat J P , Hopper J E
Published: June 2007

In yeast, galactose triggers a rapid GAL4-dependent induction of galactose/melibiose regulon (GAL/MEL) gene transcription, and glucose represses this activation. We discovered that alterations in the physical state of the GAL4 protein correlate with activation and repression of the GAL/MEL genes. Using Western immunoblot assay, we observe two electrophoretic forms of GAL4 protein-GAL4I and GAL4II-in noninduced cells. In the absence of glucose, the addition of galactose to such cells results in the rapid appearance of a third and slower-migrating form, GAL4III, which differs from at least GAL4I by phosphorylation. GAL80-deletion cells that constitutively transcribe galactose-responsive genes due to the lack of the GAL80 protein, an antagonist of the GAL4 protein, exhibit GAL4III without galactose addition. Addition of glucose, which results in rapid repression of galactose gene transcription, triggers a rapid elimination of GAL4III and an increase in GAL4II. Cycloheximide experiments provide evidence that the galactose- and glucose-triggered GAL4 protein mobility shifts are due to post-translational modification. GAL4III is labeled with [32P]phosphate in vivo; in vivo 35S-labeled GAL4III could be converted by phosphatase treatment in vitro to GAL4I. We present a model proposing that phosphorylation state changes in the GAL4 protein are key to modulating its activity.
Ultrastructural and molecular analyses of homozygous-viable Drosophila melanogaster muscle mutants indicate there is a complex pattern of myosin heavy-chain isoform distribution.
Authors: O'Donnell P T , Collier V L , Mogami K , Bernstein S I
Published: June 2007

We describe the ultrastructural and initial molecular characterization of four homozygous-viable, dominant-flightless mutants of Drosophila melanogaster. Genetic mapping indicates that these mutations are inseparable from the known muscle myosin heavy-chain (MHC) allele Mhc1, and each mutation results in a muscle-specific reduction in MHC protein accumulation. The indirect flight muscles (IFMs) of each of these homozygous mutants fail to accumulate MHC, lack thick filaments, and do not display normal cylindrical myofibrils. As opposed to the null phenotype observed in the IFM, normal amounts of MHC accumulate in the leg muscles of three of these mutants, whereas the fourth mutant shows a 45% reduction in leg muscle MHC. The ultrastructure of the tergal depressor of the trochanter muscle TDT, or jump muscle) is normal in one mutant, completely lacks thick filaments in a second mutant, and displays a reduction of thick filaments in two mutants. The thick filament reduction in this latter class of mutants is limited to the four smaller anterior cells of the TDT, indicating that the TDT is a mixed fiber-type muscle. Because all isoforms of muscle MHC are encoded by alternative splicing of transcripts from a single gene, our results suggest that there is a complex pattern of MHC isoform accumulation in Drosophila. The phenotypes of the homozygous-viable mutants provide evidence for the differential localization of MHC isoforms in different muscles, within the same muscle, and even within a single muscle cell. The mutant characteristics also suggest that the use of some alternative exons is shared among the IFM, TDT, and additional muscles whereas the use of others is unique to the IFM.