Variations in COL15A1 and COL18A1 influence age of onset of primary open angle glaucoma.

Primary open angle glaucoma (POAG) is a genetically and phenotypically complex disease that is a leading cause of blindness worldwide. Previously we completed a genome-wide scan for early-onset POAG that identified a locus on 9q22 (GLC1J). To identify potential causative variants underlying GLC1J, we used targeted DNA capture followed by high throughput sequencing of individuals from four GLC1J pedigrees, followed by Sanger sequencing to screen candidate variants in additional pedigrees. A mutation likely to cause early-onset glaucoma was not identified, however COL15A1 variants were found in the youngest affected members of 7 of 15 pedigrees with variable disease onset. In addition, the most common COL15A1 variant, R163H, influenced the age of onset in adult POAG cases. RNA in situ hybridization of mouse eyes shows that Col15a1 is expressed in the multiple ocular structures including ciliary body, astrocytes of the optic nerve and cells in the ganglion cell layer. Sanger sequencing of COL18A1, a related multiplexin collagen, identified a rare variant, A1381T, in members of three additional pedigrees with early-onset disease. These results suggest genetic variation in COL15A1 and COL18A1 can modify the age of onset of both early and late onset POAG.

Mutations associated with familial melanoma impair p16INK4 function.

Cell division is controlled by a series of positive and negative regulators which act at sequential points throughout the cell cycle. Disturbance of these checks could contribute to cancer by allowing excessive cell proliferation. The point in G1 at which cells irrevocably commit to DNA synthesis is controlled by protein complexes consisting of cyclin-dependent kinases (CDK4 or CDK6) and cyclins (D1, D2 or D3). These complexes are inhibited by low molecular weight proteins, such as p16INK4 (refs 1,2), p15INK4B (ref. 3) and p18 (ref. 4). Deletion or mutation of these CDK-inhibitors could lead to unchecked cell growth, suggesting that members of the p16INK4 family may be tumour suppressor genes. The recent detection of p16INK4 (MTS1) mutations in familial melanoma kindreds, many human tumour cell lines, and primary tumours is consistent with this idea. Previously, we described eight germline p16INK4 substitutions in 18 familial melanoma kindreds. Genetic analyses suggested that five mutations predisposed carriers to melanoma, whereas two missense mutations had no phenotypic effect. We now describe biochemical analyses of the missense germline mutations and a single somatic mutation detected in these families. Only the melanoma-predisposing mutants were impaired in their ability to inhibit the catalytic activity of the cyclin D1/CDK4 and cyclin D1/CDK6 complexes in vitro. Our data provide a biochemical rationale for the hypothesis that carriers of certain p16INK4 mutations are at increased risk of developing melanoma.

Production of transgenic calves expressing an shRNA targeting myostatin.

Myostatin (MSTN) is a well-known negative regulator of muscle growth. Animals that possess mutations within this gene display an enhanced muscling phenotype, a desirable agricultural trait. Increased neonatal morbidity is common, however, resulting from complications arising from the birth of offspring with increased fetal muscle mass. The objective of the current research was to generate an attenuated MSTN-null phenotype in a large-animal model using RNA interference to enhance muscle development without the detrimental consequences of an inactivating mutation. To this end, we identified a series of short interfering RNAs that demonstrated effective suppression of MSTN mRNA and protein levels. To produce transgenic offspring capable of stable MSTN suppression in vivo, a recombinant lentiviral vector expressing a short hairpin RNA (shRNA) targeting MSTN for silencing was introduced into bovine fetal fibroblasts. These cells were used as nucleus donors for somatic cell nuclear transfer (SCNT). Twenty blastocysts were transferred into seven recipient cows resulting in five pregnancies. One transgenic calf developed to term, but died following delivery by Caesarean-section. As an alternative strategy, microinjection of recombinant lentiviral particles into the perivitelline space of in vitro-produced bovine zygotes was utilized to produce 40 transgenic blastocysts that were transferred into 14 recipient cows, resulting in 7 pregnancies. Five transgenic calves were produced, of which three expressed the transgene. This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus, and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing.

Targeted resequencing of a genomic region influencing tameness and aggression reveals multiple signals of positive selection.

The identification of the causative genetic variants in quantitative trait loci (QTL) influencing phenotypic traits is challenging, especially in crosses between outbred strains. We have previously identified several QTL influencing tameness and aggression in a cross between two lines of wild-derived, outbred rats (Rattus norvegicus) selected for their behavior towards humans. Here, we use targeted sequence capture and massively parallel sequencing of all genes in the strongest QTL in the founder animals of the cross. We identify many novel sequence variants, several of which are potentially functionally relevant. The QTL contains several regions where either the tame or the aggressive founders contain no sequence variation, and two regions where alternative haplotypes are fixed between the founders. A re-analysis of the QTL signal showed that the causative site is likely to be fixed among the tame founder animals, but that several causative alleles may segregate among the aggressive founder animals. Using a formal test for the detection of positive selection, we find 10 putative positively selected regions, some of which are close to genes known to influence behavior. Together, these results show that the QTL is probably not caused by a single selected site, but may instead represent the joint effects of several sites that were targets of polygenic selection.

Measuring particle size distribution and mass concentration of nanoplastics and microplastics: addressing some analytical challenges in the sub-micron size range.

HYPOTHESIS: The implementation of the proposal from the European Chemical Agency (ECHA) to restrict the use of nanoplastics (NP) and microplastics (MP) in consumer products will require reliable methods to perform size and mass-based concentration measurements. Analytical challenges arise at the nanometre to micrometre interface, e.g., 800 nm-10 µm, where techniques applicable at the nanometre scale reach their upper limit of applicability and approaches applicable at the micrometre scale must be pushed to their lower limits of detection. EXPERIMENTS: Herein, we compared the performances of nine analytical techniques by measuring the particle size distribution and mass-based concentration of polystyrene mixtures containing both nano and microparticles, with the educational aim to underline applicability and limitations of each technique. FINDINGS: Light scattering-based measurements do not have the resolution to distinguish multiple populations in polydisperse samples. Nanoparticle tracking analysis (NTA), nano-flowcytometry (nFCM) and asymmetric flow field flow fractionation hyphenated with multiangle light scattering (AF4-MALS) cannot measure particles in the micrometre range. Static light scattering (SLS) is not able to accurately detect particles below 200 nm, and similarly to transmission electron microscopy (TEM) and flow cytometry (FCM), is not suitable for accurate mass-based concentration measurements. Alternatives for high-resolution sizing and concentration measurements in the size range between 60 nm and 5 µm are tunable resistive pulse sensing (TRPS) and centrifugal liquid sedimentation (CLS), that can bridge the gap between the nanometre and micrometre range.

A proinflammatory cytokine inhibits p53 tumor suppressor activity.

p53 has a key role in the negative regulation of cell proliferation, in the maintenance of genomic stability, and in the suppression of transformation and tumorigenesis. To identify novel regulators of p53, we undertook two functional screens to isolate genes which bypassed either p53-mediated growth arrest or apoptosis. In both screens, we isolated cDNAs encoding macrophage migration inhibitory factor (MIF), a cytokine that was shown previously to exert both local and systemic proinflammatory activities. Treatment with MIF overcame p53 activity in three different biological assays, and suppressed its activity as a transcriptional activator. The observation that a proinflammatory cytokine, MIF, is capable of functionally inactivating a tumor suppressor, p53, may provide a link between inflammation and tumorigenesis.

Interaction of U6 snRNA with a sequence required for function of the nematode SL RNA in trans-splicing.

Nematode trans-spliced leader (SL) RNAs are composed of two domains, an exon [the 22-nucleotide spliced leader] and a small nuclear RNA (snRNA)-like sequence. Participation in vitro of the spliced leader RNA in trans-splicing reactions is independent of the exon sequence or size and instead depends on features contained in the snRNA-like domain of the molecule. Chemical modification interference analysis has revealed that two short sequence elements in the snRNA-like domain are necessary for SL RNA activity. These elements are sufficient for such activity because when added to a 72-nucleotide fragment of a nematode U1 snRNA, this hybrid RNA could participate in trans-splicing reactions in vitro. One of the critical sequence elements may function by base-pairing with U6 snRNA, an essential U snRNA for both cis- and trans-splicing.

Argonaute2, a link between genetic and biochemical analyses of RNAi.

Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs ( approximately 22 nucleotides) derived from the double-stranded RNA trigger, but the protein components of this activity are unknown. Here, we report the biochemical purification of the RNAi effector nuclease from cultured Drosophila cells. The active fraction contains a ribonucleoprotein complex of approximately 500 kilodaltons. Protein microsequencing reveals that one constituent of this complex is a member of the Argonaute family of proteins, which are essential for gene silencing in Caenorhabditis elegans, Neurospora, and Arabidopsis. This observation begins the process of forging links between genetic analysis of RNAi from diverse organisms and the biochemical model of RNAi that is emerging from Drosophila in vitro systems.

p53-independent role of MDM2 in TGF-beta1 resistance.

Transforming growth factor-beta (TGF-beta) inhibits cell proliferation, and acquisition of TGF-beta resistance has been linked to tumorigenesis. A genetic screen was performed to identify complementary DNAs that abrogated TGF-beta sensitivity in mink lung epithelial cells. Ectopic expression of murine double minute 2 rescued TGF-beta-induced growth arrest in a p53-independent manner by interference with retinoblastoma susceptibility gene product (Rb)/E2F function. In human breast tumor cells, increased MDM2 expression levels correlated with TGF-beta resistance. Thus, MDM2 may confer TGF-beta resistance in a subset of tumors and may promote tumorigenesis by interference with two independent tumor suppressors, p53 and Rb.

Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD.

Skeletal muscle differentiation entails the coordination of muscle-specific gene expression and terminal withdrawal from the cell cycle. This cell cycle arrest in the G0 phase requires the retinoblastoma tumor suppressor protein (Rb). The function of Rb is negatively regulated by cyclin-dependent kinases (Cdks), which are controlled by Cdk inhibitors. Expression of MyoD, a skeletal muscle-specific transcriptional regulator, activated the expression of the Cdk inhibitor p21 during differentiation of murine myocytes and in nonmyogenic cells. MyoD-mediated induction of p21 did not require the tumor suppressor protein p53 and correlated with cell cycle withdrawal. Thus, MyoD may induce terminal cell cycle arrest during skeletal muscle differentiation by increasing the expression of p21.

Molecular cloning of linotte in Drosophila: a novel gene that functions in adults during associative learning.

The linotte (lio) gene was identified in a screen for mutations that disrupted 3 hr memory after olfactory associative learning, without affecting the perception of odors or electroshock. The mutagenesis yielded a transposon-tagged gene disruption, which allowed rapid cloning of genomic DNA. The lio transcription unit was identified via rescue of the lio1 learning/memory defect by induced expression of a lio+ transgene in adults. The perception of odors or electroshock remained normal when the lio+ transgene was expressed in these lio1 flies. Learning/memory remained normal when the lio+ transgene was expressed in wild-type (lio+) flies. The lio gene produces only one transcript, the level of expression of which varies throughout development. Sequence analysis indicates that lio encodes a novel protein.

RNAi Codex: a portal/database for short-hairpin RNA (shRNA) gene-silencing constructs.

Use of RNA interference (RNAi) in forward genetic screens is proliferating. Currently, short-interfering RNAs (siRNAs) and short-hairpin RNAs (shRNAs) are being used to silence genes to tease out functional information. It is becoming easier to harness RNAi to silence specific genes, owing to the development of libraries of readymade shRNA and siRNA gene-silencing constructs by using a variety of sources. RNAi Codex, which consists of a database of shRNA related information and an associated website, has been developed as a portal for publicly available shRNA resources and is accessible at http://codex.cshl.org. RNAi Codex currently holds data from the Hannon-Elledge shRNA library and allows the use of biologist-friendly gene names to access information on shRNA constructs that can silence the gene of interest. It is designed to hold user-contributed annotations and publications for each construct, as and when such data become available. We will describe features of RNAi Codex and explain the use of the tool.

Subcellular distribution of p21 and PCNA in normal and repair-deficient cells following DNA damage.

BACKGROUND: The p21 protein binds to both cyclin-dependent kinases (Cdks) and the proliferating cell nuclear antigen (PCNA). In mammalian cells, DNA damage results in an increase in the level of p53 protein, which stimulates expression of the gene encoding p21, which in turn leads to an inhibition of Cdk activity. Biochemical studies have shown that the direct interaction between p21 and PCNA blocks the latter's function in DNA replication but not in DNA repair. In addition to the p53-dependent damage response, the stimulation of quiescent cells with serum can also cause a p53-independent elevation in p21 gene expression. It is not clear, however, whether the induction of p21 protein under these two circumstances serves the same purpose. In this study, we have investigated the kinetics of p21 induction by DNA damage and serum stimulation and the consequent effects on cell-cycle progression. Using both normal and repair-deficient human cells, we have also analyzed the nuclear distribution of p21 in relation to that of PCNA. RESULTS: In vivo immunofluorescence staining experiments indicate that, following UV damage, DNA repair is not inhibited by the presence of a large amount of p21 protein in the nucleus; in contrast, cells undergoing DNA replication during S phase contain very low amounts of p21. The addition of serum induced a transitory elevation of p21 levels, whereas UV damage to cells resulted in a sustained, high level of p21 that was more tightly associated with the nuclear structure. Interestingly, cells deficient in global nucleotide excision-repair displayed a distinct pattern of detergent-insoluble p21 that co-localized with PCNA. CONCLUSIONS: The in vivo studies presented here, which are consistent with our previous findings in vitro, indicate that p21 has a differential effect on DNA replication and DNA repair, and that the induction of p21 by serum and DNA damage may have different consequences. Furthermore, the co-localization of p21 and PCNA in the nucleus of normal and repair-deficient human cells indicates that p21 and PCNA interact during post-damage events.

Accurate processing of human pre-rRNA in vitro.

We report here that the mature 5' terminus of human 18S rRNA is generated in vitro by a two-step processing reaction. In the first step, SP6 transcripts were specifically cleaved in HeLa cell nucleolar extract at three positions near the external transcribed spacer (ETS)-18S boundary. Of these cleavage sites, two were major and the other was minor. RNase T1 fingerprint and secondary nuclease analyses placed the two major cleavage sites 3 and 8 bases upstream from the mature 5' end of 18S rRNA and the minor cleavage site 1 base into the 18S sequence. All three cleavages yielded 5'-hydroxyl, 2'-3'-cyclic phosphate termini and were 5' of adenosine residues in the sequence UACCU, which was repeated three times near the ETS-18S boundary. In the second step, the initial cleavage product containing 3 bases of ETS was converted to an RNA with a 5' terminus identical to that of mature 18S RNA by an activity found in HeLa cell cytoplasmic extracts.

Loss-of-function genetics in mammalian cells: the p53 tumor suppressor model.

Using an improved system for the functional identification of active antisense fragments, we have isolated antisense fragments which inactivate the p53 tumour suppressor gene. These antisense fragments map in two small regions between nt 350 and 700 and nt 800 and 950 of the coding sequence. These antisense fragments appear to act by inhibition of p53 mRNA translation both in vivo and in vitro. Expression of these antisense fragments overcame the p53-induced growth arrest in a cell line which expresses a thermolabile mutant of p53 and extended the in vitro lifespan of primary mouse embryonic fibroblasts. Continued expression of the p53 antisense fragment contributed to immortalisation of primary mouse fibroblasts. Subsequent elimination of the antisense fragment in these immortalised cells led to restoration of p53 expression and growth arrest, indicating that immortal cells continuously require inactivation of p53. Expression of MDM2 or SV40 large T antigen, but not E7 nor oncogenic ras, overcomes the arrest induced by restoration of p53 expression. Functional inactivation of both p21 and bax (by overexpression of Bcl2), but not either alone, allowed some bypass of p53-induced growth arrest, indicating that multiple transcriptional targets of p53 may mediate its antiproliferative action. The ability to conditionally inactivate and subsequently restore normal gene function may be extremely valuable for genetic analysis of genes for which loss-of-function is involved in specific phenotypes.

Deletion of the p16 and p15 genes in human bladder tumors.

BACKGROUND: Two genes, p16 (also known as CDKN2, INK4A, or MTS1) and p15 (also described as INK4B or MTS2), are found in tandem at chromosome 9p21. These genes are designated as candidate tumor suppressor genes because they encode proteins that function as negative cell cycle regulators. (The encoded polypeptides inactivate specific cyclin-protein kinase complexes that are required for progression through the cell cycle.) Molecular genetic studies have revealed that deletion of the p16 and p15 genes occurs frequently in cancer cell lines and in certain malignant neoplasms. PURPOSE: We evaluated the frequency of p16 and p15 gene alterations in a well-characterized cohort of human transitional cell bladder cancers, and we explored potential associations between alterations in these genes and tumor stage and/or grade. METHODS: Tumor tissue and normal tissue from 110 patients with transitional cell carcinoma of the urinary bladder were examined. The status of the p16 and p15 genes in these tissues was determined by Southern blotting and hybridization with gene-specific probes, by coupled polymerase chain reaction and single-strand conformation polymorphism analysis (PCR-SSCP), and by sequencing DNA fragments produced during PCR. Associations between alterations in the genes and tumor stage and/or grade were evaluated using the two-tailed Fisher's exact test. RESULTS: Homozygous deletion (both alleles lost) of the p16 and the p15 genes was observed in 11 and nine bladder tumors, respectively. Eight of the 11 tumors exhibiting complete loss of the p16 gene also displayed homozygous deletion of the p15 gene. Exclusive loss of either gene was detected in only three tumors. Hemizygous deletion (one allele lost, also referred to as loss of heterozygosity [LOH] of the p16 and/or p15 genes was observed in eight tumors. Rearrangement of the two genes was indicated in three additional tumors. No point mutations were identified in either gene. The overall frequency of alteration in this cohort of bladder tumors was approximately 18% for each gene (in 20 [18.3%, 95% confidence interval (CI) = 11.1%-25.6%] of 109 informative tumors for the p16 gene and in 18 [18%, 95% CI = 10.5%-25.5%] of 100 informative tumors for the p15 gene). A statistically significant association between p16 gene alteration and bladder tumors of low stage (P < .01) and grade (P < .01) was observed; a significant association between p15 gene alteration and tumors of low stage (P < .01) was also detected. CONCLUSIONS: Alteration of the p16 and p15 genes, especially coincident homozygous deletion, appears to be a common event in bladder cancer.

Mutations in the p16INK4/MTS1/CDKN2, p15INK4B/MTS2, and p18 genes in primary and metastatic lung cancer.

We examined the genomic status of cyclin-dependent kinase-4 and -6 inhibitors, p16INK4,p15INK4B, and p18, in 40 primary lung cancers and 31 metastatic lung cancers. Alterations of the p16INK4 gene were detected in 6 (2 insertions and 4 homozygous deletions) of 22 metastatic non-small cell lung cancers (NSCLCs; 27%), but none were detected in 25 primary NSCLCs, 15 primary small cell lung cancers (SCLCs), or 9 metastatic SCLCs, indicating that mutation in the p16INK4 gene is a late event in NSCLC carcinogenesis. Although three intragenic mutations of the p15INK4B gene were detected in 25 primary NSCLCs (12%) and five homozygous deletions of the p15INK4B gene were detected in 22 NSCLCs (23%), no genetic alterations of the p15INK4B gene were found in primary and metastatic SCLCs. The p18 gene was wild type in these 71 lung cancers, except 1 metastatic NSCLC which showed loss of heterozygosity. We also examined alterations of these three genes and expression of p16INK4 in 21 human lung cancer cell lines. Alterations of the p16INK4 and p15INK4B genes were detected in 71% of the NSCLC cell lines (n = 14) and 50% of the NSCLC cell lines (n = 14), respectively, but there were none in the 7 SCLC cell lines studied. No p18 mutations were detected in these 21 cell lines. These results indicate that both p16INK4 and p15INK4B gene mutations are associated with tumor progression of a subset of NSCLC, but not of SCLC, and that p15INK4B mutations might also be an early event in the molecular pathogenesis of a subset of NSCLC.

Cloning and characterization of murine p16INK4a and p15INK4b genes.

Progression through the G1 phase of the cell cycle is regulated in part by the D-type cyclin-dependent kinases, cdk4 and cdk6. Genes encoding two specific inhibitors of these kinases, human p16(INK4a/MTS1) and p15(INK4b/MTS2), map to a region of common cytogenetic abnormalities on chromosome 9p21. The murine cognates of these genes were isolated and identified as mouse p16INK4a and p15INK4b based on their homology to their human counterparts and their selective transcriptional induction by SV40T-antigen and TGF-beta, respectively. Both genes map to position C3-C6 on mouse chromosome 4, in a region syntenic with human chromosome 9p. Amplification of polyadenylated mRNA by polymerase chain reactions revealed no expression of mouse p16INK4a in many normal tissues, whereas p15INK4b was expressed ubiquitously. Like human p16INK4a, mouse p16INK4a binds specifically to cdk4 and cdk6 in vitro and inhibits the phosphorylation of the retinoblastoma protein, pRb, by each of these cyclin D-dependent kinases. In mouse MEL erythroleukemia cells, p16INK4a associates preferentially with cdk6 under conditions where cdk4 and cdk6 are coexpressed at equivalent levels. Expression vectors encoding human or mouse p16INK4a caused G1 phase arrest in NIH3T3 fibroblasts, and cyclin D1- and cdk4-dependent pRb kinase activities were inhibited in the p16INK4a-arrested cells.

Accurate and efficient RNA polymerase III transcription in a cell-free extract prepared from Ascaris suum embryos.

High-speed supernatant (S100) extracts derived from homogenized Ascaris suum embryos efficiently transcribe added RNA polymerase III templates including cloned 5S rRNA genes of the filarial parasite Brugia malayi. Several criteria, including two-dimensional RNase T1 oligonucleotide fingerprint analysis, indicate that in vitro transcription is accurately initiated and terminated.