Understanding and modulating apoptosis in industrial cell culture.

Currently, the production of therapeutic recombinant proteins relies heavily on the large-scale culture of eukaryotic cells that secrete the protein of interest into the media. It has been recognized that programmed cell death, or apoptosis, may pose a significant hurdle to maximum productivity in such systems. With a greater understanding of the molecular events causing apoptosis, alterations can be made to the cells and culture conditions to prevent apoptosis and enhance volumetric productivity.

Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis.

GATA-3 is one member of a growing family of related transcription factors which share a strongly conserved expression pattern in all vertebrate organisms. In order to elucidate GATA-3 function using a direct genetic approach, we have disrupted the murine gene by homologous recombination in embryonic stem cells. Mice heterozygous for the GATA3 mutation are fertile and appear in all respects to be normal, whereas homozygous mutant embryos die between days 11 and 12 postcoitum (p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver haematopoiesis.

Embryonic expression and cloning of the murine GATA-3 gene.

We describe the embryonic expression pattern as well as the cloning and initial transcriptional regulatory analysis of the murine (m) GATA-3 gene. In situ hybridization shows that mGATA-3 mRNA accumulation is temporally and spatially regulated during early development: although found most abundantly in the placenta prior to 10 days of embryogenesis, mGATA-3 expression becomes restricted to specific cells within the embryonic central nervous system (in the mesencephalon, diencephalon, pons and inner ear) later in gestation. GATA-3 also shows a restricted expression pattern in the peripheral nervous system, including terminally differentiating cells in the cranial and sympathetic ganglia. In addition to this distinct pattern in the nervous system, mGATA-3 is also expressed in the embryonic kidney and the thymic rudiment, and further analysis showed that it is expressed throughout T lymphocyte differentiation. To begin to investigate how this complex gene expression pattern is elicited, cloning and transcriptional regulatory analyses of the mGATA-3 gene were initiated. At least two regulatory elements (one positive and one negative) appear to be required for appropriate tissue-restricted regulation after transfection of mGATA-3-directed reporter genes into cells that naturally express GATA-3 (T lymphocytes and neuroblastoma cells). Furthermore, this same region of the locus confers developmentally appropriate expression in transgenic mice, but only in a subset of the tissues that naturally express the gene.

Temporal and spatial changes in GATA transcription factor expression are coincident with development of the chicken optic tectum.

The molecular mechanisms specifying patterns of gene expression in the vertebrate brain, which in turn determine the developmental fates of specific neurons, are yet to be clearly defined. Individual members of a recently identified family of transcriptional regulatory proteins, the GATA factors, are required for the differentiation of certain hematopoietic cell lineages. We show here that two of the members of this gene family, GATA-2 and GATA-3, are expressed within discrete cell populations of the chicken optic tectum during embryogenesis, and that they have highly restricted patterns of expression in the developing chicken brain. Furthermore, the induction of GATA factor expression within specific cell layers parallels the well established spatial (rostral to caudal) and temporal pattern of optic tectum development. The observation that both the timing of appearance and the localization of expression of GATA-2 and GATA-3 are correlated with optic tectum development suggest that these transcription factors may be associated with the initiation of gene transcription required for the determination of specific neuronal fates within visual areas of the vertebrate brain.

A GATA family transcription factor is expressed along the embryonic dorsoventral axis in Drosophila melanogaster.

The GATA transcription factors are a family of C4 zinc finger-motif DNA-binding proteins that play defined roles in hematopoiesis as well as presumptive roles in other tissues where they are expressed (e.g., testis, neuronal and placental trophoblast cells) during vertebrate development. To investigate the possibility that GATA proteins may also be involved in Drosophila development, we have isolated and characterized a gene (dGATAa) encoding a factor that is quite similar to mammalian GATA factors. The dGATAa protein sequence contains the two zinc finger DNA-binding domain of the GATA class but bears no additional sequence similarity to any of the vertebrate GATA factors. Analysis of dGATAa gene transcription during Drosophila development revealed that its mRNA is expressed at high levels during early embryogenesis, with transcripts first appearing in the dorsal portion of the embryo just after cellularization. As development progresses, dGATAa mRNA is present at high levels in the dorsal epidermis, suggesting that dGATAa may be involved in determining dorsal cell fate. The pattern of expression in a variety of dorsoventral polarity mutants indicates that dGATAa lies downstream of the zygotic patterning genes decapentaplegic and zerknüllt.

Quantitation of RNA using the polymerase chain reaction.

Sequential use of reverse transcriptase and the polymerase chain reaction (RT-PCR) permits rapid and sensitive detection of specific RNAs. However, the greatest advantage of RT-PCR, its remarkable sensitivity, has also limited its usefulness in quantitative applications, since the effects of minor variations in reaction conditions from sample to sample are greatly magnified during the amplification process. Several recently developed techniques circumvent this problem, allowing accurate quantitation of RNA using RT-PCR.

Expression of the chicken GATA factor family during early erythroid development and differentiation.

The DNA motif WGATAR has been identified within transcriptional regulatory domains of globin and other erythroid-specific genes and the activator proteins that bind to this regulatory element, the GATA factors, belong to a multi-gene family that is expressed in chicken erythroid cells. Here we show that, as in chickens, multiple members of the GATA factor family are expressed in human and murine erythroid cells. During the early stages of chicken embryogenesis (well before blood island formation), each of the GATA family members is transcribed with a unique temporal and spatial pattern. In the primitive erythroid lineage, transcription of the embryonic epsilon-globin gene parallels GATA-1 expression while the switch to beta-globin transcription in definitive erythroid cells is directly preceded by a pronounced increase in GATA-3 accumulation. The timing and pattern of expression of these different mRNAs during avian erythroid development and differentiation suggests that temporally regulated changes in GATA factor expression are required for vertebrate hematopoiesis.

cis and trans regulation of tissue-specific transcription.

Analysis of both the cis-regulatory sequences which control globin gene switching as well as the trans-acting factors which bind to these sequences to elicit a differential, developmentally regulated response has lent insight into the general mechanisms responsible for tissue-specific gene regulation. We show here that the chicken adult beta-globin gene promoter sequences are intimately involved in competitive interaction with the beta/epsilon-globin enhancer to regulate differentially epsilon- versus beta-globin gene transcription. Secondly, we show that the family of GATA transcription factors directs gene regulation in a variety of discrete cell types, and describe potential cellular target genes for each member of the GATA factor family, as well as potential mechanisms whereby multiple GATA factors expressed in a single cell might be used to elicit differential transcriptional activities.

Evidence for torsional stress in transcriptionally activated chromatin.

The existence of torsional stress in eukaryotic chromatin has been controversial. To determine whether it could be detected, we probed the structure of an alternating AT tract. These sequences adopt cruciform geometry when the DNA helix is torsionally strained by negative supercoiling. The single-strand-specific nuclease P1 was used to determine the structure of an alternating AT sequence upstream of the Xenopus beta-globin gene when assembled into chromatin in microinjected Xenopus oocytes. The pattern of cleavage by P1 nuclease strongly suggests that the DNA in this chromatin template is under torsional stress. The cruciform was detected specifically in the most fully reconstituted templates at later stages of chromatin assembly, suggesting that negative supercoiling is associated with chromatin maturation. Furthermore, the number of torsionally strained templates increased dramatically at the time when transcription of assembled chromatin templates began. Transcription itself has been shown to induce supercoiling, but the requisite negative supercoiling for cruciform extrusion by (AT)n in oocytes was not generated in this way since the characteristic P1 cutting pattern was retained even when RNA polymerase elongation was blocked with alpha-amanitin. Thus, torsional stress is associated with transcriptional activation of chromatin templates in the absence of ongoing transcription.

Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor delta gene enhancer.

A family of transcriptional activators has recently been identified in chickens; these transcriptional activators recognize a common consensus motif (WGATAR) through a conserved C4 zinc finger DNA-binding domain. One of the members of this multigene family, cGATA-3, is most abundantly expressed in the T-lymphocyte cell lineage. Analysis of human and murine GATA-3 factors shows a striking degree of amino acid sequence identity and similar patterns of tissue specificity of expression in these three organisms. The murine and human factors are abundantly expressed in a variety of human and murine T-cell lines and can activate transcription through a tissue-specific GATA-binding site identified within the human T-cell receptor delta gene enhancer. We infer that the murine and human GATA-3 proteins play a central and highly conserved role in vertebrate T-cell-specific transcriptional regulation.

Primer extension analysis of mRNA isolated from transfected cell lines.

Primer extension is a relatively quick and convenient means by which transcription from a gene transfected into tissue-culture cells can be monitored. The technique can be used to accurately determine the site of transcription initiation or to quantify the amount of cap-site-specific message produced.

Activity and tissue-specific expression of the transcription factor NF-E1 multigene family.

NF-E1, a DNA-binding protein that recognizes the general consensus motif WGATAR, is the first tissue-specific factor to be identified in erythroid cells. Using a probe from the murine GF-1 (NF-E1) cDNA clone, we isolated three homologous chicken cDNAs: One of these corresponds to an mRNA (NF-E1a) that is abundantly and exclusively expressed in erythroid cells; a second mRNA (NF-E1b) is also expressed in all developmental stages of erythroid cells but is additionally found in a limited subset of other chicken tissues; mRNA representative of a third gene (NF-E1c) is expressed only in definitive (adult) erythrocytes within the red cell lineage but is also abundantly expressed in T lymphocytes and brain. All NF-E1 proteins are highly conserved within the DNA-binding domain and bind to the consensus motif with similar affinities in vitro; they are also all stimulatory trans-acting factors in vivo. The factors differ quantitatively in their ability to trans-activate reporter genes in which the number and position of cognate binding sites is varied relative to the transcriptional initiation site. These data suggest that the NF-E1 consensus motif directs a broader and more complicated array of developmental transcriptional regulatory processes than has been assumed and that NF-E1c may play a unique regulatory role in the developing chicken brain and in T lymphocytes.

Lemon juice, sunlight, and tattoos.

In folklore, tattoos are thought to fade if rubbed with lemon juice and exposed to sunlight. The authors tested this hypothesis on shaved, tattooed rats. Tattooed controls were used. Additional substances and conditions were included in the study (tretinoin gel, liquid nitrogen, and dermabrasion). The authors conclude that India ink tattoos do not fade significantly with sunlight and lemon juice nor with the application of tretinoin gel, liquid nitrogen, or combinations thereof.