Cosmids from the Vollmer-Yanofsky library identified with a chromosome VII probe.

In microorganisms, genes can often be cloned directly by complementation of mutants with a genomic library

Internal translation initiation in the mRNA for the Neurospora crassa albino-3 gene

The "ribosome scanning model" for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of multiple translational start sites on the mRNA of the Neurospora crassa gene albino-3 (al-3), encoding the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthase. This was revealed by the molecular analysis of an al-3 mutant carrying a deletion within the coding sequence, which was expected to prevent the synthesis of a functional geranylgeranyl-pyrophosphate synthase because of ribosome frameshifting and premature translational termination. However, the mutants could maintain appreciable geranylgeranyl-pyrophosphate synthase activity through a mechanism operating at the translational level, whereby a fraction of ribosomes initiated protein synthesis from either of two internal in-frame AUG codons located downstream of the deletion, thus producing a shortened but still active version of the geranylgeranyl-pyrophosphate synthase. The results presented indicate that the internal AUG codons are recognized mainly or solely by direct ribosome binding rather than by "leaky scanning" from the 5' end of the mRNA.The "ribosome scanning model" for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of multiple translational start sites on the mRNA of the Neurospora crassa gene albino-3 (al-3), encoding the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthase. This was revealed by the molecular analysis of an al-3 mutant carrying a deletion within the coding sequence, which was expected to prevent the synthesis of a functional geranylgeranyl-pyrophosphate synthase because of ribosome frameshifting and premature translational termination. However, the mutants could maintain appreciable geranylgeranyl-pyrophosphate synthase activity through a mechanism operating at the translational level, whereby a fraction of ribosomes initiated protein synthesis from either of two internal in-frame AUG codons located downstream of the deletion, thus producing a shortened but still active version of the geranylgeranyl-pyrophosphate synthase. The results presented indicate that the internal AUG codons are recognized mainly or solely by direct ribosome binding rather than by "leaky scanning" from the 5' end of the mRNA

CBM1, a Neurospora crassa genomic cosmid library in pAC3 and its use for walking on the right arm of linkage group VII

Gene cloning in Neurospora crassa is often achieved by mutant complementation. However, the cloning strategy sometimes requires the isolation of a specific genomic region (by chromosome walking) before transformation of N. crassa. This is the case, for example, if the gene to be isolated has a non-selectable phenotype. Here we specifically describe the construction of the cosmid vector, pAC3, which is designed for direct transformation of N. crassa, its utilization for the construction of a genomic library, and chromosome walking in the region of un-10 on linkage group VII

Human cortical organoids expose a differential function of GSK3 on cortical neurogenesis

The regulation of the proliferation and polarity of neural progenitors is crucial for the development of the brain cortex. Animal studies have implicated glycogen synthase kinase 3 (GSK3) as a pivotal regulator of both proliferation and polarity, yet the functional relevance of its signaling for the unique features of human corticogenesis remains to be elucidated. We harnessed human cortical brain organoids to probe the longitudinal impact of GSK3 inhibition through multiple developmental stages. Chronic GSK3 inhibition increased the proliferation of neural progenitors and caused massive derangement of cortical tissue architecture. Single-cell transcriptome profiling revealed a direct impact on early neurogenesis and uncovered a selective role of GSK3 in the regulation of glutamatergic lineages and outer radial glia output. Our dissection of the GSK3-dependent transcriptional network in human corticogenesis underscores the robustness of the programs determining neuronal identity independent of tissue architecture

Achlya mitochondrial DNA: gene localization and analysis of inverted repeats

Mitochondrial DNA from four strains of the oomycete Achlya has been compared and nine gene loci mapped, including that of the ribosomal protein gene, var1 . Examination of the restriction enzyme site maps showed the presence of four insertions relative to a map common to all four strains. All the insertions were found in close proximity to genic regions. The four strains also cotained the inverted repeat first observed in A. ambisexualis (Hudspeth et al. 1983), allowing an examination by analysis of retained restriction sites of the evolutionary stability of repeated DNA sequences relative to single copy sequences. Although the inverted repeat is significantly more stable than single copy sequences, more detailed analysis indicated that this stability is limited to the portion encoding the ribosomal RNA genes. Thus, the apparent evolutionary stability of the repeat does not appear to derive from the inverted repeat structure per se.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47563/1/438_2004_Article_BF00330510.pd

The Expression of Vasoactive Intestinal Peptide Receptor 1 Is Negatively Modulated by MicroRNA 525-5p

Background: The human Vasoactive Intestinal Peptide (VIP) is a neurokine with effects on the immune system where it is involved in promoting tolerance. In this context, one of its receptors, VPAC1, has been found to be down-modulated in cells of the immune network in response to activating stimuli. In particular, the bacterial liposaccaride (LPS), a strong activator of the innate immune system, induces a rapid decrease of VPAC1 expression in monocytes and this event correlates with polymorphisms in the 3'-UTR of the gene. Methodology/Principal Findings: MicroRNA 525-5p, having as putative target the 3'-UTR region of VPAC1, has been analysed for its expression in monocytes and for its role in down-modulating VPAC1 expression. We report here that miR-525-5p is promptly up-regulated in LPS-treated monocytes. This microRNA, when co-transfected in 293T cells together with a construct containing the 3'-UTR of the VPAC1 gene, significantly reduced the luciferase activity in a standard expression assay. The U937 cell line as well as primary monocytes enforced to express miR-525-5p, both down-modulate VPAC1 expression at similar extent. Conclusions/Significance: Our results show that the response to an inflammatory stimulus elicits in monocytes a rapid increase of miR-525-5p that targets a signaling pathway involved in the control of the immune homeostasis

Composition-based statistics and translated nucleotide searches: Improving the TBLASTN module of BLAST

BACKGROUND: TBLASTN is a mode of operation for BLAST that aligns protein sequences to a nucleotide database translated in all six frames. We present the first description of the modern implementation of TBLASTN, focusing on new techniques that were used to implement composition-based statistics for translated nucleotide searches. Composition-based statistics use the composition of the sequences being aligned to generate more accurate E-values, which allows for a more accurate distinction between true and false matches. Until recently, composition-based statistics were available only for protein-protein searches. They are now available as a command line option for recent versions of TBLASTN and as an option for TBLASTN on the NCBI BLAST web server. RESULTS: We evaluate the statistical and retrieval accuracy of the E-values reported by a baseline version of TBLASTN and by two variants that use different types of composition-based statistics. To test the statistical accuracy of TBLASTN, we ran 1000 searches using scrambled proteins from the mouse genome and a database of human chromosomes. To test retrieval accuracy, we modernize and adapt to translated searches a test set previously used to evaluate the retrieval accuracy of protein-protein searches. We show that composition-based statistics greatly improve the statistical accuracy of TBLASTN, at a small cost to the retrieval accuracy. CONCLUSION: TBLASTN is widely used, as it is common to wish to compare proteins to chromosomes or to libraries of mRNAs. Composition-based statistics improve the statistical accuracy, and therefore the reliability, of TBLASTN results. The algorithms used by TBLASTN are not widely known, and some of the most important are reported here. The data used to test TBLASTN are available for download and may be useful in other studies of translated search algorithms