Characterization of specific cDNA background synthesis introduced by reverse transcription in RT-PCR assays.

To block expression of NMDA receptor NR1 subunit, we injected into rat hippocampus a Herpes Simplex Virus type 1 derived vector bearing a sequence for NR1 antisense. RT-PCR assays with RNA from hippocampus of animals injected either with NR1 antisense vector, control vector or vehicle, showed an amplification signal compatible with NR1 antisense which could be attributed either to an endogenous NR1 antisense or to an artifact. RT-PCR was performed either with different primers or without primers in the RT, using RNA from different tissues. RNAse protection assay was carried out to characterize the amplified signal nature. Our results show that the template for the unexpected amplified fragment was NR1 mRNA currently expressed in nervous tissue. We considered this basal amplification of a mRNA in a RT-PCR assay as "background amplification". After background subtraction, a significant signal only remained when samples from NR1 antisense vector injected animals were used, demonstrating that this was the only source for NR1 antisense. Background amplification at RT in the absence of primers, can constitute a troubling factor in quantitative nucleic acid determination, leading to major interference, particularly when both sense and antisense sequences are present in the sample. Since RT introduced a significant background signal for every gene analyzed, we propose that RT must be always performed also without primers. Then, this signal should be identified, quantified and subtracted from the specific reaction amplification signal.

Chromatin and alternative splicing.

Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing.

Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing.

Here we investigate the promoter control of alternative splicing by studying two transcriptional activators on templates under replicating conditions. SV40 large T-antigen (T-Ag) activates template replication only 2-fold but transcription 25-fold. T-Ag-mediated replication, reported to inhibit RNA polymerase II elongation, provokes a 10- to 30-fold increase in the inclusion of the fibronectin EDI exon into mature mRNA. The T-Ag effect is exon specific, occurs in cis and depends strictly on DNA replication and not on cell transformation. VP16, an activator of transcriptional initiation and elongation, has a similar effect on transcription but the opposite effect on splicing: EDI inclusion is inhibited by 35-fold. VP16 completely reverts the T-Ag effect, but a VP16 mutant with reduced elongation ability provokes only partial reversion. Both T-Ag and VP16 promote conspicuous co-localization of mRNA with nuclear speckles that contain the SR protein SF2/ASF, a positive regulator of EDI inclusion. Therefore, we conclude that co-localization of transcripts and speckles is not sufficient to stimulate EDI inclusion.

Coordination between transcription and pre-mRNA processing.

A large body of work has proved that transcription by RNA polymerase II and pre-mRNA processing are coordinated events within the cell nucleus. Capping, splicing and polyadenylation occur while transcription proceeds, suggesting that RNA polymerase II plays a role in the regulation of these events. The presence and degree of phosphorylation of the carboxy-terminal domain of RNA polymerase II large subunit is important for functioning of the capping enzymes, the assembly of spliceosomes and the binding of the cleavage/polyadenylation complex. Nuclear architecture and gene promoter structure have also been shown to play key roles in coupling between transcription and splicing.

MRNA encoding a putative RNA helicase of the DEAD-box gene family is up-regulated in trypomastigotes of Trypanosoma cruzi.

Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands.

[Molecular biology and medicine at the end of the 20th Century]. / Biologia molecular y medicina a fines del siglo XX.

This paper reviews basic concepts of modern molecular biology with the premise that its influence in today's medicine is so important that its knowledge cannot remain limited to a few experts. I first analyze the overall structure and organization of human genes, their split nature and the flow of genetic information from DNA to protein. The role of transcriptional control in the regulation of gene expression and cell differentiation is described by introducing experimental examples that define the importance of "master" genes. Basic concepts of genetic engineering, the generation of transgenic and knock out animals and the uses of molecular biology in clinical diagnosis, paternity tests and forensic medicine are presented. Finally, I discuss the possibilities of gene therapy and the fantasies and realities of transgenesis and cloning by nuclear transplant in humans.

A novel frameshift mutation (2436insT) produces an immediate stop codon in the autosomal dominant polycystic kidney disease 2 (PKD2) gene.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder that can be caused by mutations in at least three different genes. Several mutations have been identified in PKD1 and PKD2 genes. Most of the mutations found in PKD2 gene are predicted to cause premature termination of the protein. METHODS: We analysed an Argentinian family characterized previously as PKD2. The PKD2 gene was amplified from genomic DNA using 17 primer pairs and the products were analysed by heteroduplex analysis. PCR products that showed a variation by heteroduplex analysis were sequenced directly. The mutation was confirmed by sequencing relatives. The segregation of the mutation in this family was verified by restriction endonuclease digestion of PCR products obtained from genomic DNA of all family members. Results and conclusions. Here, we report a novel mutation present in an Argentinian family characterized as PKD2 by linkage analysis. The mutation, shared by all affected members of the family, is a thymidine insertion at position 2436 of the gene, which results in a translation frameshift and creates an immediate stop codon. This mutation is expected to lead to a truncated protein that lacks the interacting domain with the PKD1 gene product. The thymidine insertion abolished a Ddel restriction site, allowing a rapid test for detection of PKD2 carriers in the family.

Biologia molecular y medicina a fines del siglo XX. / [Molecular biology and medicine at the end of the 20th Century]

This paper reviews basic concepts of modern molecular biology with the premise that its influence in todays medicine is so important that its knowledge cannot remain limited to a few experts. I first analyze the overall structure and organization of human genes, their split nature and the flow of genetic information from DNA to protein. The role of transcriptional control in the regulation of gene expression and cell differentiation is described by introducing experimental examples that define the importance of [quot ]master[quot ] genes. Basic concepts of genetic engineering, the generation of transgenic and knock out animals and the uses of molecular biology in clinical diagnosis, paternity tests and forensic medicine are presented. Finally, I discuss the possibilities of gene therapy and the fantasies and realities of transgenesis and cloning by nuclear transplant in humans.

Interaction between the (-170) CRE and the (-150) CCAAT box is necessaryfor efficient activation of the fibronectin gene promoter by cAMP and ATF-2.

The fibronectin promoter contains an ATF/cyclic AMP (cAMP) response element (CRE) site two helical turns upstream of a CCAAT site with which it interacts. We investigated the effects of mutating these (-170) CRE and(-150) CCAAT elements on the promoter activity regulated by three different modulators previously known to act through CRE: ATF-2, cAMP and E1a. While the cooperation seems to play no role in E1a action, integrity of the (-150) CCAAT is necessary for ATF-2 and cAMP efficient activation in a cell-specific manner. These results show that the CRE and CCAAT elements function as a 'composite element' and establish a cell-specific function for CRE-CCAAT synergy.

Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer.

Alternative mRNA splicing of the fibronectin EDI exon is controlled by a purine-rich exonic splicing enhancer (ESE), postulated as a binding site for SR proteins. By using a transient expression alternative splicing assay combined with promoter swapping, we have demonstrated that the promoter can also control EDI splicing, arguing for coupling between the transcription and splicing machineries. We now report that the SR proteins SF2/ASF and 9G8 stimulate EDI splicing in vivo and that their effect requires an intact EDI ESE. Most importantly, we show that sensitivity to these SR proteins critically depends on the promoter structure, suggesting that the transcription machinery modulates their recruitment to the ESE.

[Autosomal dominant polycystic kidney disease: detection of a new mutation in the PKD1 gene]. / Poliquistosis renal autosómica dominante. Detección de una nueva mutación en el gen PKD1.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder characterized by genetic heterogeneity. Up to three loci are involved in this disease, PKD1 on chromosome 16p13.3, PKD2 on 4q21, and a third locus of unknown location. Since the identification of the PKD1 gene, the interest was centered in the characterization of the mutations responsible for the disease. Most mutations found were diverse and situated throughout the gene with no phenotypic correlation. Here we describe a new mutation in exon 44 from PKD1 gene in a family previously characterized as PKD1 by linkage analysis. The mutation is a single base substitution from a C to a T at position 12220 originating a stop codon at the mutation site. This would lead to premature termination and the formation of a truncated protein lacking part of the carboxi-terminus.

Novel processing in a mammalian nuclear 28S pre-rRNA: tissue-specific elimination of an 'intron' bearing a hidden break site.

Splitting and apparent splicing of ribosomal RNA, both previously unknown in vertebrates, were found in rodents of the genus Ctenomys. Instead of being formed by a single molecule of 4.4 kb, 28S rRNA is split in two molecules of 2.6 and 1.8 kb. A hidden break, mapping within a 106 bp 'intron' located in the D6 divergent region, is expressed in mature ribosomes of liver, lung, heart and spleen, as well as in primary fibroblast cultures. Testis-specific processing eliminates the intron and concomitantly the break site, producing non-split 28S rRNA molecules exclusively in this organ. The intron is flanked by two 9 bp direct repeats, revealing the acquisition by insertion of a novel rRNA processing strategy in the evolution of higher organisms.

Evidence for a role of the alternatively spliced ED-I sequence of fibronectin during ovarian follicular development.

This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor messenger RNA, play specific roles during development of the ovarian follicle. In particular, we were interested in determining the effect of the ED-I (also termed ED-A) type III repeat, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I+ FN, whereas total FN levels remained relatively constant. ED-I+ FN levels were higher in small follicles, corresponding to the phase of granulosa cell proliferation. The hypothesis of a physiological role for ED-I+ FN was further supported by the finding of a regulation of the alternative splicing of FN in primary cultures of bovine granulosa cells by factors known to control ovarian follicular development. cAMP produced a 10-fold decrease in the relative proportion of the ED-I region. In contrast, transforming growth factor-beta elicited a 2-fold stimulation of overall FN synthesis and a 4-fold increase in the synthesis of ED-I containing FN. This effect was evident at the protein (Western blots) and messenger RNA (Northern blots) levels. Although a negative correlation (P < 0.001) was detected between ED-I+ FN and estradiol levels in follicular fluid, this steroid was unable to modulate in vitro the alternative splicing of FN. A possible mitogenic effect of ED-I+ FN was suggested by the observation that a recombinant peptide corresponding to the ED-I domain stimulated DNA synthesis in a bovine granulosa cell line (BGC-1), whereas a peptide corresponding to the flanking type III sequences had no effect. The hypothesis of ED-I+ FN as a growth regulatory factor was further strengthened by the fact that depletion of FN from BGC-1-conditioned medium, which contained ED-I+ FN, abrogated its mitogenic activity, whereas plasma FN was without effect. We propose that changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.

Isolated polycystic liver disease not linked to polycystic kidney disease 1 and 2.

Autosomal dominant polycystic liver disease occurs commonly in association with autosomal dominant polycystic kidney disease, types 1 and 2. It may also exist as a separate entity, genetically distinct from autosomal dominant polycystic kidney disease types 1 and 2, as has been recently established to exist in a Belgian family. We report here a large Argentinian family of Spanish-Belgian ancestry with autosomal dominant polycystic liver disease, where proximal and distal markers for both polycystic kidney disease 1 and 2 failed to demonstrate genetic linkage. The data support the notion that polycystic liver disease and autosomal dominant polycystic kidney disease may have separate chromosomal loci.

Poliquistosis renal autosómica dominante. Detección de una nueva mutación en el gen PKD1. / [Autosomal dominant polycystic kidney disease: detection of a new mutation in the PKD1 gene]

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder characterized by genetic heterogeneity. Up to three loci are involved in this disease, PKD1 on chromosome 16p13.3, PKD2 on 4q21, and a third locus of unknown location. Since the identification of the PKD1 gene, the interest was centered in the characterization of the mutations responsible for the disease. Most mutations found were diverse and situated throughout the gene with no phenotypic correlation. Here we describe a new mutation in exon 44 from PKD1 gene in a family previously characterized as PKD1 by linkage analysis. The mutation is a single base substitution from a C to a T at position 12220 originating a stop codon at the mutation site. This would lead to premature termination and the formation of a truncated protein lacking part of the carboxi-terminus.

Expression of RGD minus fibronectin that does not form extracellular matrix fibrils is sufficient to decrease tumor metastasis.

Fibronectin (FN) is a plasma and extracellular matrix (ECM) glycoprotein, the expression of which may modulate the invasive and metastatic abilities of cancer cells. LMM3 is a cell line derived from the highly metastatic mouse mammary adenocarcinoma MM3 and is unable to express FN both at protein and mRNA levels. To study the role of FN in the metastatic process, LMM3 cells were stably transfected with 2 variants (wt and RGD-minus) of a full length human FN cDNA. All analyzed clones secreted recombinant FN and although none assembled FN in the ECM they showed an in vitro reduced migratory ability and an increased adhesive capacity. FN-producing cells were assayed for experimental and spontaneous metastasis. All clones tested showed a significant reduction in the number of experimental lung metastasis when compared with a control clone. Similar trends were observed for spontaneous metastatic ability. Our results indicate that the expression of FN that lacks the well-recognized RGD cell-binding site and that does not form ECM fibrils, is sufficient to decrease the metastatic potential of cancer cells. Our results also suggest that an RGD-independent mechanism may be acting in the prevention of metastasis.

Cloning of a cdc2-related protein kinase from Trypanosoma cruzi that interacts with mammalian cyclins.

Two cdc2-related protein kinases (crk), tzcrk3 and tzcrk1, from the protozoan parasite Trypanosoma cruzi were cloned. tzcrk3 encodes a 35 kDa protein sharing 51.5% amino acid identity with human cdc2 and 82% identity with Trypanosoma brucei CRK3. tzcrk1 encodes a 33 kDa protein sharing 52.7% identity with human cdc2 and a high degree of identity (> 78%) with T. brucei CRK1, Leishmania mexicana CRK1 and Trypanosoma congolense CRK1. A recombinant TzCRK1 protein was able to phosphorylate histone HI and retinoblastoma protein. Western blotting using a polyclonal antibody raised against the recombinant TzCRK1 protein showed that the kinase is present in all life cycle stages of the parasite. A PSTAIRE antiserum detected proteins of 32, 33 and 35 kDa, with differential expression in the life cycle of the parasite. Transfection of COS-7 cells with tzcrk1 demonstrated for the first time that a CRK protein can bind mammalian cyclins; TzCRK1 co-immunoprecipitated with cyclins E, D3 and A suggesting a role for this kinase in cell cycle control. These results indicate that T. cruzi might have cyclin homologues that control the activity of the CRK proteins and that a complex mechanism would exist in order to regulate the kinases involved in the cell cycle and the differentiation processes of the parasite.

Downregulation of fibronectin transcription in highly metastatic adenocarcinoma cells.

Silencing of fibronectin (FN) expression seems to be one of the key mechanisms underlying metastatic behaviour. An inverse correlation exists between FN expression levels and the metastatic potential of two related murine mammary adenocarcinomas, M3 and MM3. Primary cultures of M3 tumour, which is moderately metastatic to lung (40% incidence), show a conspicuous FN extracellular matrix (ECM) and high levels of FN mRNA, while primary cultures of the highly metastatic MM3 tumour (95% lung incidence) are negative for FN in immunofluorescence and show at least 40-fold lower levels of FN mRNA, only detectable by RT-PCR, with a different pattern of alternatively spliced EDI isoforms compared to M3 cells. We show that the FN promoter sequence is not altered in MM3 cells. Transfection experiments with CAT constructs indicate that silencing occurs at the transcriptional level, involving the 220-bp proximal promoter region.