A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells

A transgenic mouse model of metastatic prostate cancer has been developed that is 100% penetrant in multiple pedigrees. Nucleotides −6500 to +34 of the mouse cryptdin-2 gene were used to direct expression of simian virus 40 T antigen to a subset of neuroendocrine cells in all lobes of the FVB/N mouse prostate. Transgene expression is initiated between 7 and 8 weeks of age and leads to development of prostatic intraepithelial neoplasia within a week. Prostatic intraepithelial neoplasia progresses rapidly to local invasion. Metastases to lymph nodes, liver, lung, and bone are common by 6 months. Tumorigenesis is not dependent on androgens. This model indicates that the neuroendocrine cell lineage of the prostate is exquisitely sensitive to transformation and provides insights about the significance of neuroendocrine differentiation in human prostate cancer.

Aldehyde dehydrogenase class 3 expression: Identification of a cornea-preferred gene promoter in transgenic mice

Aldehyde dehydrogenase class 3 (ALDH3) constitutes 20–40% of the total water-soluble proteins in the mammalian cornea. Here, we show by Northern blot analysis that ALDH3 expression in the mouse is at least 500-fold higher in the cornea than in any other tissue examined, with very low levels of expression detected in the stomach, urinary bladder, ocular lens, and lung. Histochemical localization reveals that this exceptional level of expression in the mouse cornea occurs in the anterior epithelial cells and that little ALDH3 is present in the keratocytes or corneal endothelial cells. A 13-kbp mouse ALDH3 promoter fragment containing >12 kbp of the 5′ flanking sequence, the 40-bp untranslated first exon, and 29 bp of intron 1 directed cat reporter gene expression to tissues that express the endogenous ALDH3 gene, except that transgene promoter activity was higher in the stomach and bladder than in the cornea. By contrast, when driven by a 4.4-kbp mouse ALDH3 promoter fragment [1,050-bp 5′ flanking region, exon 1, intron 1 (3.4 kbp), and 7 bp of exon 2] expression of the cat reporter gene was confined to the corneal epithelial cells, except for very low levels in the liver, effectively reproducing the corneal expression pattern of the endogenous ALDH3 gene. These results indicate that tissue-specific expression of ALDH3 is determined by positive and negative elements in the 5′ flanking region of the gene and suggests putative silencers located in intron 1. We demonstrate regulatory sequences capable of directing cornea-specific gene expression, affording the opportunity for genetic engineering in this transparent tissue.

A link between DNA methylation and epigenetic silencing in transgenic Volvox carteri

Epigenetic silencing of foreign genes introduced into plants poses an unsolved problem for transgenic technology. Here we have used the simple multicellular green alga Volvox carteri as a model to analyse the relation of DNA methylation to transgenic silencing. Volvox DNA contains on average 1.1% 5-methylcytosine and 0.3% N6-methyladenine, as revealed by electrospray mass spectrometry and phosphoimaging of chromatographically separated 32P-labelled nucleotides. In two nuclear transformants of V.carteri, produced in 1993 by biolistic bombardment with a foreign arylsulphatase gene (C-ars), the transgene is still expressed in one (Hill 181), but not in the other (Hill 183), after an estimated 500–1000 generations. Each transformant clone contains multiple intact copies of C-ars, most of them integrated into the genome as tandem repeats. When the bisulphite genomic sequencing protocol was applied to examine two select regions of transgenic C-ars, we found that the inactivated copies (Hill 183) exhibited a high-level methylation (40%) of CpG dinucleotides, whereas the active copies (Hill 181) displayed low-level (7%) CpG methylation. These are average values from 40 PCR clones sequenced from each DNA strand in the two portions of C-ars. The observed correlation of CpG methylation and transgene inactivation in a green alga will be discussed in the light of transcriptional silencing.

Estimation of divergence times from multiprotein sequences for a few mammalian species and several distantly related organisms

When many protein sequences are available for estimating the time of divergence between two species, it is customary to estimate the time for each protein separately and then use the average for all proteins as the final estimate. However, it can be shown that this estimate generally has an upward bias, and that an unbiased estimate is obtained by using distances based on concatenated sequences. We have shown that two concatenation-based distances, i.e., average gamma distance weighted with sequence length (d2) and multiprotein gamma distance (d3), generally give more satisfactory results than other concatenation-based distances. Using these two distance measures for 104 protein sequences, we estimated the time of divergence between mice and rats to be approximately 33 million years ago. Similarly, the time of divergence between humans and rodents was estimated to be approximately 96 million years ago. We also investigated the dependency of time estimates on statistical methods and various assumptions made by using sequence data from eubacteria, protists, plants, fungi, and animals. Our best estimates of the times of divergence between eubacteria and eukaryotes, between protists and other eukaryotes, and between plants, fungi, and animals were 3, 1.7, and 1.3 billion years ago, respectively. However, estimates of ancient divergence times are subject to a substantial amount of error caused by uncertainty of the molecular clock, horizontal gene transfer, errors in sequence alignments, etc.

Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: A useful model to monitor germ cells in a live vertebrate

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange–red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3′ region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20–25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

Spontaneous retinopathy in HLA-A29 transgenic mice

Humans who have inherited the class I major histocompatibility allele HLA-A29 have a markedly increased relative risk of developing the eye disease termed birdshot chorioretinopathy. This disease affecting adults is characterized by symmetrically scattered, small, cream-colored spots in the fundus associated with retinal vasculopathy and inflammatory signs causing damage to the ocular structures, leading regularly to visual loss. To investigate the role of HLA-A29 in this disease, we introduced the HLA-A29 gene into mice. Aging HLA-A29 transgenic mice spontaneously developed retinopathy, showing a striking resemblance to the HLA-A29-associated chorioretinopathy. These results strongly suggest that HLA-A29 is involved in the pathogenesis of this disease. Elucidation of the role of HLA-A29 should be assisted by this transgenic model.

Galactose-extended glycans of antibodies produced by transgenic plants

Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that β1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human β1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal β1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal β1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human β1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.

L-selectin can facilitate metastasis to lymph nodes in a transgenic mouse model of carcinogenesis

L-selectin mediates homing of lymphocytes to lymph nodes (LN). Transgenic mice that express rat insulin promoter regulated simian virus 40 Tag (RIP-Tag) develop large, local cancers that metastasize to liver but not LN. To test whether this lack of LN metastases reflects their absence from the circulation, transgenic mice were produced that express Tag (T), L-selectin (L), and Escherichia coli LacZ (Z), in pancreatic β cells. LTZ mice developed insulinomas that specifically had LN metastases; metastasis was blocked by an anti L-selectin mAb. LacZ+ tumor cells from these LN homed to secondary LN upon transfer. These results suggest that the highly vascularized islet carcinomas are shedding tumor cells into the bloodstream, which is a necessary but insufficient condition for metastasis to occur; L-selectin can facilitate homing of such tumor cells to LN, resulting in metastasis.

Galanin transgenic mice display cognitive and neurochemical deficits characteristic of Alzheimer's disease

Galanin is a neuropeptide with multiple inhibitory actions on neurotransmission and memory. In Alzheimer's disease (AD), increased galanin-containing fibers hyperinnervate cholinergic neurons within the basal forebrain in association with a decline in cognition. We generated transgenic mice (GAL-tg) that overexpress galanin under the control of the dopamine β-hydroxylase promoter to study the neurochemical and behavioral sequelae of a mouse model of galanin overexpression in AD. Overexpression of galanin was associated with a reduction in the number of identifiable neurons producing acetylcholine in the horizontal limb of the diagonal band. Behavioral phenotyping indicated that GAL-tgs displayed normal general health and sensory and motor abilities; however, GAL-tg mice showed selective performance deficits on the Morris spatial navigational task and the social transmission of food preference olfactory memory test. These results suggest that elevated expression of galanin contributes to the neurochemical and cognitive impairments characteristic of AD.

Trypanosome spliced leader RNA genes contain the first identified RNA polymerase II gene promoter in these organisms

Typical general transcription factors, such as TATA binding protein and TFII B, have not yet been identified in any member of the Trypanosomatidae family of parasitic protozoa. Interestingly, mRNA coding genes do not appear to have discrete transcriptional start sites, although in most cases they require an RNA polymerase that has the biochemical properties of eukaryotic RNA polymerase II. A discrete transcription initiation site may not be necessary for mRNA synthesis since the sequences upstream of each transcribed coding region are trimmed from the nascent transcript when a short m7G-capped RNA is added during mRNA maturation. This short 39 nt m7G-capped RNA, the spliced leader (SL) sequence, is expressed as an ∼100 nt long RNA from a set of reiterated, though independently transcribed, genes in the trypanosome genome. Punctuation of the 5′ end of mRNAs by a m7G cap-containing spliced leader is a developing theme in the lower eukaryotic world; organisms as diverse as Euglena and nematode worms, including Caenorhabditis elegans, utilize SL RNA in their mRNA maturation programs. Towards understanding the coordination of SL RNA and mRNA expression in trypanosomes, we have begun by characterizing SL RNA gene expression in the model trypanosome Leptomonas seymouri. Using a homologous in vitro transcription system, we demonstrate in this study that the SL RNA is transcribed by RNA polymerase II. During SL RNA transcription, accurate initiation is determined by an initiator element with a loose consensus of CYAC/AYR(+1). This element, as well as two additional basal promoter elements, is divergent in sequence from the basal transcription elements seen in other eukaryotic gene promoters. We show here that the in vitro transcription extract contains a binding activity that is specific for the initiator element and thus may participate in recruiting RNA polymerase II to the SL RNA gene promoter.

Posttranscriptional Gene Silencing in Transgenic Sugarcane. Dissection of Homology-Dependent Virus Resistance in a Monocot That Has a Complex Polyploid Genome1

RNA-mediated, posttranscriptional gene silencing has been determined as the molecular mechanism underlying transgenic virus resistance in many plant virus-dicot host plant systems. In this paper we show that transgenic virus resistance in sugarcane (Saccharum spp. hybrid) is based on posttranscriptional gene silencing. The resistance is derived from an untranslatable form of the sorghum mosaic potyvirus strain SCH coat protein (CP) gene. Transgenic sugarcane plants challenged with sorghum mosaic potyvirus strain SCH had phenotypes that ranged from fully susceptible to completely resistant, and a recovery phenotype was also observed. Clones derived from the same transformation event or obtained after vegetative propagation could display different levels of virus resistance, suggesting the involvement of a quantitative component in the resistance response. Most resistant plants displayed low or undetectable steady-state CP transgene mRNA levels, although nuclear transcription rates were high. Increased DNA methylation was observed in the transcribed region of the CP transgenes in most of these plants. Collectively, these characteristics indicate that an RNA-mediated, homology-dependent mechanism is at the base of the virus resistance. This work extends posttranscriptional gene silencing and homology-dependent virus resistance, so far observed only in dicots, to an agronomically important, polyploid monocot.

Protective Function of Chloroplast 2-Cysteine Peroxiredoxin in Photosynthesis. Evidence from Transgenic Arabidopsis1

2-Cysteine peroxiredoxins (2-CPs) constitute a ubiquitous group of peroxidases that reduce cell-toxic alkyl hydroperoxides to their corresponding alcohols. Recently, we cloned 2-CP cDNAs from plants and characterized them as chloroplast proteins. To elucidate the physiological function of the 2-CP in plant metabolism, we generated antisense mutants in Arabidopsis. In the mutant lines a 2-CP deficiency developed during early leaf and plant development and eventually the protein accumulated to wild-type levels. In young mutants with reduced amounts of 2-CP, photosynthesis was impaired and the levels of D1 protein, the light-harvesting protein complex associated with photosystem II, chloroplast ATP synthase, and ribulose-1,5-bisphosphate carboxylase/oxygenase were decreased. Photoinhibition was particularly pronounced after the application of the protein synthesis inhibitor, lincomycin. We concluded that the photosynthetic machinery needs high levels of 2-CP during leaf development to protect it from oxidative damage and that the damage is reduced by the accumulation of 2-CP protein, by the de novo synthesis and replacement of damaged proteins, and by the induction of other antioxidant defenses in 2-CP mutants.

Characterization and Subcellular Compartmentation of Recombinant 4-Hydroxyphenylpyruvate Dioxygenase from Arabidopsis in Transgenic Tobacco1

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.