Aimee H. Bakken

Simultaneous Expression of hCNT1-CFP and hENT1-YFP in Madin-Darby Canine Kidney Cells LOCALIZATION AND VECTORIAL TRANSPORT STUDIES

To test the hypothesis that human concentrative and equilibrative nucleoside transporters (hCNT1 and hENT1) are present on the apical and basolateral membrane, respectively, we constructed a Madin-Darby canine kidney (MDCK) cell line that simultaneously and stably expresses recombinant hCNT1 and hENT1 gene products tagged with CFP and YFP fluorescent proteins, respectively. Using a confocal microscope, both hCNT1-CFP and hENT1-YFP were found to be distributed uniformly on the plasma membrane of undifferentiated MDCK cells. Upon differentiation of the MDCK cells on Transwell filter inserts, hCNT1-CFP was visualized exclusively on the apical membrane, whereas hENT1-YFP appeared predominantly on the basolateral membrane. As differentiation proceeded, there was an increase in alkaline phosphatase activity, and activity of hENT1 in the apical compartment decreased while hCNT1 activity remained constant. These results suggest that, on differentiation, hENT1 is sorted to the basolateral membrane. This was confirmed when the hCNT1-mediated uptake of [3H]uridine from the apical compartment of the differentiated cells was found to be ∼20-fold higher and that for hENT1 was ∼4-fold lower than the corresponding uptake from the basal compartment. As observed in vivo, the net transport of [3H]adenosine was from the apical to the basal compartment, whereas that for 14C-deoxyadenosine was from the basal to the apical compartment. In summary, we have shown for the first time that hCNT1 and hENT1 are expressed in polarized MDCK cells on the apical and basolateral membrane, respectively, allowing vectorial transport in both directions depending on the relative activity (ratio of maximal transporter activity to affinity) of each transporter for their substrates.

Patterns of RNA synthesis in early meiotic prophase oocytes from fetal mouse ovaries

In a study of the early meiotic prophase stages of mouse oogenesis from d12 of gestation to 10d post-partum the patterns of RNA synthesis during these stages of oogenesis using H3-uridine incorporation as visualized by light microscope autoradiography are reported. We find that chromosomal RNA synthesis occurs in all stages except early to mid-pachytene, the time of maximum chromosome condensation. Diplotene and dictyate nuclei are the most heavily labelled stages. Nucleolar labelling ceases before leptotene and reappears in late pachytene or early diplotene, even though nucleoli can be identified in all stages except early to mid-pachytene.

Functional expression of human intestinal Na+-dependent and Na+-independent nucleoside transporters in Xenopus laevis oocytes☆

We have shown previously that the human jejunal brush border membrane expresses both the N1 (cif) and the N2 (cit) Na+-dependent (concentrative) nucleoside transporters but not the Na+-independent (facilitative) nitrobenzylmercaptopurineriboside (NBMPR)-sensitive (es) transporter (Patil SD and Unadkat JD, Am J Physiol, 272: 1314-1320, 1997). In the present study, we have demonstrated that when Xenopus laevis oocytes are microinjected with human jejunal mRNA, four nucleoside transporters are expressed simultaneously, namely the N1 and N2 Na+-dependent nucleoside transporters and the es and the NBMPR-insensitive (ei) Na+-independent transporters. The expressed Na+-dependent nucleoside transporters showed substrate specificity identical to that previously described by us using jejunal brush border membrane vesicles (Patil SD and Unadkat JD, Am J Physiol, 272: 1314-1320, 1997). The expressed es and ei Na+-independent transporters demonstrated broad substrate selectivity with both purines and pyrimidines capable of inhibiting the uptake of guanosine and thymidine mediated by this transporter. The expressed Na+-dependent nucleoside transporters mediated the transport of their respective nucleoside substrates with a high affinity and a low capacity, whereas the es and the ei transporters mediated the transport of nucleosides with a low affinity and a high capacity. Collectively, these observations suggest that the Na+-independent nucleoside transporters are expressed in the basolateral membrane of the human jejunal epithelium. Based on these data, we hypothesize that the concentrative transporters in the brush border membrane and equilibrative transporters in the basolateral membrane are arranged in series in the human jejunal epithelium to allow efficient vectorial transport of nucleosides from the lumen to the blood. The simultaneous expression of four nucleoside transporters in X. laevis oocytes establishes a basis for molecular cloning of these four human nucleoside transporters.

RNA-protein interactions of stored 5S RNA with TFIIIA and ribosomal protein L5 during Xenopus oogenesis☆

We studied the pathway of 5S RNA during oogenesis in Xenopus laevis from its storage in the cytoplasm to accumulation in the nucleus, the sequence requirements for the 5S RNA to follow that pathway, and the 5S RNA-protein interactions that occur during the mobilization of stored 5S RNA for assembly into ribosomes. In situ hybridization to sections of oocytes indicates that 5S RNA first becomes associated with the amplified nucleoli during vitellogenesis when the nucleoli are activity synthesizing ribosomal RNA and assembling ribosomes. When labeled 5S RNA is microinjected into the cytoplasm of stage V oocytes, it migrates into the nucleus, whether microinjected naked or complexed with the protein TFIIIA as a 7S RNP storage particle. During vitellogenesis, a nonribosome bound pool of 5S RNA complexed with ribosomal protein L5 (5S RNPs) is formed, which is present throughout the remainder of oogenesis. Immunoprecipitation assays on homogenates of microinjected oocytes showed that labeled 5S RNA can become complexed either with L5 or with TFIIIA. Nucleotides 11 through 108 of the 5S RNA molecule provide the necessary sequence and conformational information required for the formation of immunologically detectable complexes with TFIIIA or L5 and for nuclear accumulation. Furthermore, labeled 5S RNA from microinjected 7S RNPs can subsequently become associated with L5. Such labeled 5S RNA is found in both 5S RNPs and 7S RNPs in the cytoplasm, but only in 5S RNPs in the nucleus of microinjected oocytes. These data suggest that during oogenesis a major pathway for incorporation of 5S RNA into nascent ribosomes involves the migration of 5S RNA from the nucleus to the cytoplasm for storage in an RNP complex with TFIIIA, exchange of that protein association for binding with ribosomal protein L5, and a return to the nucleus for incorporation into ribosomes as they are being assembled in the amplified nucleoli.

A quantitative electron microscopic analysis of transcription in sea urchin embryos

In an effort to define stage-specific embryonic patterns of sea urchin transcription, we have examined by electron microscopy the distribution of nascent RNP fibrils in dispersed chromatin from nuclei of Strongylocentrotus purpuratus gastrulae. Detailed analysis of individual embryonic nuclei has revealed several new features of nuclear RNA production. Most (82%) of the active chromatin regions observed were represented by only a single fibril. 11% of the active regions contained multiple RNP fibril arrays with an average RNA polymerase density of 1.7±1.0 polymerases/μm of chromatin and an average contour length of 4.7±2.8 μm chromatin. An analysis of the lengths of RNP fibrils in single vs. multiple fibril arrays indicates that the differential distribution of RNA polymerases is due to different rates of initiation rather than to different lengths of transcription units (assuming the rate of RNA chain elongation to be constant). We discuss these data in relation to various transcriptional parameters measured by other workers and to EM analyses of other embryonic nuclei. Elucidation of transcriptional patterns in gastrula embryos can provide the basis for further comparative studies of transcription at other stages of sea urchin development in which rates of total genomic transcription vary but the rate at individual loci is as yet unknown.

Human intestinal es nucleoside transporter: molecular characterization and nucleoside inhibitory profiles

Purpose: To clone and sequence the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter (es) from the human small intestine and to examine the capacities of nucleosides and nucleoside analogs to inhibit the uptake of uridine by this transporter. Methods: Using PCR, es was cloned from a cDNA library of the human small intestine. The uptake of 3H-uridine (10 μM) by the recombinant es, expressed in Xenopus oocytes, was measured in the presence (2 mM) and absence of nucleosides and nucleoside analogs. Results: The amino acid sequence of this es transporter was identical to that of the human placental es transporter. Uptake of 3H-uridine by this es transporter was inhibitable by 1 μM NBMPR. Removal of the oxygen from the 3′ position or from both the 2′ and 3′ positions, but not from 2′ or 5′ position, resulted in a partial or total loss of the capacity of the nucleosides to inhibit 3H-uridine uptake. No modifications of the adenosine base or of the uridine base (except for 3 and 6 positions on uracil) affected nucleoside inhibitory capacity. Conclusions: The es transporters of the human intestine and placenta are identical in their amino acid sequences. Moreover, the inhibitory profiles of various nucleoside analogs in inhibiting the uptake of uridine by the intestinal es transporter are similar to those obtained with the as-yet-uncloned human erythrocyte es transporter. Collectively, these findings suggest that the es transporter does not appear to be functionally variant in the human placenta, small intestine or erythrocytes.

Transcription in developing sea urchins: Electron microscopic analysis of cleavage, gastrula and prism stages

Nuclei from three different developmental stages of Strongylocentrotus purpuratus were examined for stage-specific differences in transcription. After dispersal in hypotonic solution and preparation for electron microscopic (EM) observation, nuclei from cleavage, gastrula, and prism stages all displayed beaded chromatin with similar numbers of nucleosomes per μm chromatin. The DNA/chromatin packing ratios computed from these nucleosome frequencies are 1.9–2.0 for both transcribed and nontranscribed chromatin. Several stage-specific differences were also observed. (1) Prism stage nuclei exhibited a smooth, nonbeaded chromatin configuration within the active ribosomal RNA genes (packing ratio=1.1 μm DNA/1.0 μm chromatin). (2) The rapid cell cycle of cleavage stage embryos produced some nuclei with metaphase chromosomes and some with DNA replication intermediates. (3) The average lengths of the nascent nonribosomal transcripts were comparable in gastrula and prism nuclei but shorter in cleavage nuclei. (4) And, the cleavage stage chromatin had a class of short, repeated ribonucleoprotein (RNP) fibril arrays that were not observed at any other developmental stage. This special class consisted of long stretches of chromatin (50–160 μm) covered with clusters of very short RNP fibrils (length=0.04±0.02 μm). These repeating clusters ranged in length from 0.1–0.2 μm chromatin. The characteristics of these arrays suggest they are active histone genes. If so, then this is the first report of the EM morphology of transcribing histone genes. These putative histone genes contributed significantly to the amount of transcription observed in cleavage nuclei while the number and distribution of all other RNP fibrils were similar on cleavage and gastrula chromatin. These data are discussed in relation to reported changes in the RNA synthetic rates of sea urchin embryos.

Transcription of Xenopus borealis rRNA genes in nuclei of Xenopus laevis oocytes.

Abstract Recombinant plasmids that contained a single rRNA gene and spacer from Xenopus borealis were injected into the nuclei of Xenopus laevis oocytes. After spreading the contents of injected nuclei for electron microscopy, we found transcription units indicative of the accurate transcription of the borealis gene. Both the number of RNA polymerase molecules per transcription unit and the total number of active genes per nucleus were similar to those found for injected laevis genes. We also injected laevis oocytes with a plasmid that contained both a shortened laevis rRNA gene and a full-length borealis rRNA gene. Visualization of transcription on this plasmid showed that usually either a short or a long transcription unit was present on any one molecule. The morphologies of the short and long transcription units were consistent with their representing transcription of the laevis and borealis genes, respectively. Both types of transcription unit were present in roughly equal numbers. Since the laevis oocyte transcribed injected borealis rRNA genes as efficiently as it transcribed injected laevis rRNA genes, it stands in marked contrast to the situation in embryos of interspecific hybrids between laevis and borealis where transcription occurs only from the laevis rRNA genes. Possible explanations for the difference in transcriptional behavior of the borealis rRNA gene in hybrid embryos and laevis oocytes are discussed.

Views on the nature of the gene, the structure and function of the chromosome, and heterochromatic heredity.

Goldschmidt’s early acceptance of the classical definition of genes, as discrete, hereditary units, linearly arranged on chromosomes (derived from the Mendelian genetic analyses of the Morgan school) changed eventually to outright rejection1,2 when he considered the facts regarding position effects discovered by Sturtevant in 19253 in which physical rearrangements among the chromosomes lead to different activities of the same gene. He proposed an alternative model in 1938 (see below) in which the chromosome was the basic functional unit of heredity and any breakage or rearrangement within the chromosome would result in a change in the genetic functioning of the chain-like molecule as a whole.