The Arabidopsis ARGOS-LIKE gene regulates cell expansion during organ growth

Cell expansion, and its coordination with cell division, plays a critical role in the growth and development of plant organs. However, the genes controlling cell expansion during organogenesis are largely unknown. Here, we demonstrate that a novel Arabidopsis gene, ARGOS-LIKE (ARL), which has some sequence homology to the ARGOS gene, is involved in this process. Reduced expression or overexpression of ARL in Arabidopsis results in smaller or larger cotyledons and leaves as well as other lateral organs, respectively. Anatomical examination of cotyledons and leaves in ARL transgenic plants demonstrates that the alteration in size can be attributed to changes in cell size rather than cell number, indicating that ARL plays a role in cell expansion-dependent organ growth. ARL is upregulated by brassinosteroid (BR) and this induction is impaired in the BR-insensitive mutant bri1, but not in the BR-deficient mutant det2. Ectopic expression of ARL in bri1-119 partially restores cell growth in cotyledons and leaves. Our results suggest that ARL acts downstream of BRI1 and partially mediates BR-related cell expansion signals during organ growth.     

Suppression of a cold-sensitive mutation in ribosomal protein S5 reveals a role for RimJ in ribosome biogenesis

A specific mutation of Escherichia coli ribosomal protein S5, in which glycine is changed to aspartate at position 28 [S5(G28D)], results in cold sensitivity and defects in ribosome biogenesis and translational fidelity. In an attempt to understand the roles of S5 in these essential cellular functions, we selected extragenic suppressors and identified rimJ as a high-copy suppressor of the cold-sensitive phenotype associated with the S5(G28D) mutation. Our studies indicate that RimJ overexpression suppresses the growth defects, anomalous ribosome profiles and mRNA misreading exhibited by the S5(G28D) mutant strain. Although previously characterized as the N -acetyltransferase of S5, our data indicate that RimJ, when devoid of acetyltransferase activity, can suppress S5(G28D) defects thus indicating that the suppression activity of RimJ is not dependent on its acetyltransferase activity. Additionally, RimJ appears to associate with pre-30S subunits indicating that it acts on the ribonucleoprotein particle. These findings suggest that RimJ has evolved dual functionality; it functions in r-protein acetylation and as a ribosome assembly factor in E. coli .     

A 3' splice site mutation in a nuclear gene encoding a mitochondrial ribosomal protein in Neurospora crassa

We showed previously that the cyt-21+ gene of Neurospora crassa encodes a mitochondrial ribosomal protein homologous to Escherichia coli ribosomal protein S-16 (Kuiper, M. T. R., Akins, R. A., Holtrop, M., de Vries, H., and Lambowitz, A. M. (1988) J. Biol. Chem. 263, 2840-2847). A mutation in this gene, cyt-21-1, results in deficiency of mitochondrial small ribosomal subunits and small rRNA (Collins, R. A., Bertrand, H., LaPolla, R. J., and Lambowitz, A. M. (1979) Mol. Gen. Genet. 177, 73-84). In the present work, cloning and sequencing of the cyt-21-1 mutant allele show that it contains a single dG to dA transition at the 3' splice site AG of the first intron in the protein coding region. This mutation leads to inactivation of the normal 3' splice site and activation of a cryptic 3' splice site, 15 nucleotides downstream. The use of this cryptic splice site results in an in-frame deletion of 5 amino acids from the cyt-21 protein. Comparison of mutant and wild-type mitochondrial small ribosomal subunit proteins showed one protein, S-24, with an altered electrophoretic mobility, consistent with the predicted deletion. The mutant ribosomal protein is still capable of binding to mitochondrial small ribosomal subunits, but results in abnormal mitochondrial ribosome assembly.     

Regulation of the cell expansion gene RHD3 during Arabidopsis development

The RHD3 (ROOT HAIR DEFECTIVE3) gene encodes a putative GTP-binding protein required for appropriate cell enlargement in Arabidopsis. To obtain insight into the mechanisms of RHD3 regulation, we conducted a molecular genetic dissection of RHD3 gene expression and function. Gene fusion and complementation studies show that the RHD3 gene is highly expressed throughout Arabidopsis development and is controlled by two major regulatory regions. One regulatory region is located between -1,500 and -600 bp upstream of the RHD3 gene and is required for vascular tissue expression. The other region is intragenically located and includes the 558-bp first intron, which is responsible for high-level expression of RHD3 throughout the plant. The presence and location of this intron is essential for gene function because constructs lacking this intron or constructs with the intron in an abnormal position are unable to functionally complement the rhd3 mutations. We also analyzed the role of other RHD genes and the plant hormones auxin and ethylene in RHD3 regulation, and we determined that these act downstream or independently from the RHD3 pathway. This study shows that multiple levels of regulation are employed to ensure the appropriate expression of RHD3 throughout Arabidopsis development.     

The Drosophila ribosomal protein L14-encoding gene, identified by a novel Minute mutation in a dense cluster of previously undescribed genes in cytogenetic region 66D

The Minute phenotype results from mutations at?>50 loci scattered throughout the genome of Drosophila. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. Here, we report a novel P-element induced Minute mutation, P{lacW}M(3)66D ¹ , that maps to region 66D on chromosome 3L. Flies heterozygous for P{lacW}M(3)66D ¹ have a strong Minute phenotype. Molecular characterisation of the chromosomal region revealed three previously undescribed Drosophila genes clustered within a 5-kb genomic fragment. Two of the genes have significant sequence homology to genes for the mammalian ribosomal proteins L14 and RD, respectively, and share a joint 240-bp promoter region harbouring the P-element insert. Quantitative Northern blot analyses showed the mutation to affect RPL14 mRNA levels only. Interestingly, the reduction in abundance of RPL14 mRNA is not constitutive, indicating that the promoter function abolished by the inserted P-element is utilised with different efficiencies in different developmental situations. Remobilisation of the P element produced wild-type flies with normal levels of RPL14 mRNA, demonstrating that the mutant phenotype is caused by the insertion. P{lacW}M(3)66D ¹ joins a growing list of Minute mutations associated with ribosomal protein-haploinsufficiency.     

The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division

Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.     

The intron-containing L3 ribosomal protein gene (RPL3): sequence analysis and identification of U43 and of two novel intronic small nucleolar RNAs

Isolation and sequencing of bovine and human intron-containing L3 ribosomal protein genes are here reported. They exhibit very similar organisation, both comprising 10 exons and nine introns. A polymorphic locus, involving a 19-bp deletion, was found in intron 6 of the human gene. The frequency of the two alleles has been estimated in 200 haploid genomes. In bovine and human genes intron sequences are rather different, except for limited regions, located in corresponding positions, which show a surprisingly high degree of identity. All these regions contain conserved features defining the box C/D class of small nucleolar RNAs. Demonstration is given that U43 small nucleolar RNA is encoded within the first intron of both bovine and human genes. Single nucleotide sequences, encoding two novel species of small nucleolar RNAs (U82, U83a and U83b), are located in introns 3, 5 and 7. Their expression has been investigated and a possible role of these molecules in 2'-O-ribose methylation of rRNAs is discussed.     

The Drosophila ribosomal protein L14-encoding gene, identified by a novel Minute mutation in a dense cluster of previously undescribed genes in cytogenetic region 66D

The Minute phenotype results from mutations at >50 loci scattered throughout the genome of Drosophila. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. Here, we report a novel P-element induced Minute mutation, P{lacW}M(3)66D ¹ , that maps to region 66D on chromosome 3L. Flies heterozygous for P{lacW}M(3)66D ¹ have a strong Minute phenotype. Molecular characterisation of the chromosomal region revealed three previously undescribed Drosophila genes clustered within a 5-kb genomic fragment. Two of the genes have significant sequence homology to genes for the mammalian ribosomal proteins L14 and RD, respectively, and share a joint 240-bp promoter region harbouring the P-element insert. Quantitative Northern blot analyses showed the mutation to affect RPL14 mRNA levels only. Interestingly, the reduction in abundance of RPL14 mRNA is not constitutive, indicating that the promoter function abolished by the inserted P-element is utilised with different efficiencies in different developmental situations. Remobilisation of the P element produced wild-type flies with normal levels of RPL14 mRNA, demonstrating that the mutant phenotype is caused by the insertion. P{lacW}M(3)66D ¹ joins a growing list of Minute mutations associated with ribosomal protein-haploinsufficiency. Received: 20 January 1997 / Accepted: March 3 1997     

A critical role of lambda 5 protein in B cell development.

The lambda 5 gene is a homolog of immunoglobulin J lambda-C lambda genes, expressed specifically in immature B-lineage cells. Lambda 5-encoded molecules form membrane complexes with mu or D mu proteins in association with an additional protein specifically expressed in immature B cells that is encoded by the Vpre-B gene. We have generated mice in which the lambda 5 gene is inactivated by targeted gene disruption in embryonic stem cells. In these mice, B cell development in the bone marrow is blocked at the pre-B cell stage. However, the blockade is leaky, allowing B cells to populate the peripheral immune system at a low rate. These cells are allelically excluded and able to respond to antigen.     

CDK5RAP3, an essential regulator of checkpoint,interacts with RPL26 and maintains the stability of cell growth

Purpose and MaterialsCDK5RAP3 (CDK5 regulatory subunit associated protein 3) was originally identified as a binding protein of CDK5. It is a crucial gene controlling biological functions, such as cell proliferation, apoptosis, invasion, and metastasis. Although previous studies have also shown that CDK5RAP3 is involved in a variety of signalling pathways, however, the mechanism of CDK5RAP3 remains largely undefined. This study utilized MEFs from conditional knockout mice to inhibit CDK5RAP3 and knockdown CDK5RAP3 in MCF7 to explore the role of CDK5RAP3 in cell growth, mitosis, and cell death.ResultsCDK5RAP3 was found to be widely distributed throughout the centrosome, spindle, and endoplasmic reticulum, indicating that it is involved in regulating a variety of cellular activities. CDK5RAP3 deficiency resulted in instability of cell growth. CDK5RAP3 deficiency partly blocks the cell cycle in G₂/M by downregulating CDK1 (Cyclin‐dependent kinase 1) and CCNB1 (Cyclin B1) expression levels. The cell proliferation rate was decreased, thereby slowing down the cell growth rate. Furthermore, the results showed that CDK5RAP3 interacts with RPL26 (ribosome protein L26) to regulate the mTOR pathway. CDK5RAP3 and RPL26 deficiency inhibited mTOR/p‐mTOR protein and induce autophagy, resulting in an upregulation of the percentage of apoptosis, and the upregulated percentage of apoptosis also slowed cell growth.ConclusionsOur experiments show that CDK5RAP3 interacts with RPL26 and maintains the stability of cell growth. It shows that CDK5RAP3 plays an important role in cell growth and can be used as the target of gene medicine.

In normal, CDK5RAP3 is distributed in the centrosome, spindle and endoplasmic reticulum, the cells undergoes the growth and proliferation. However, when CDK5RAP3 is deficient, the cell cycle is blocked in G₂/M and cell proliferation slows down, and the partial cycle block does not cause apoptosis. Additionally, CDK5RAP3 distributed in the endoplasmic reticulum combined with the deficiency of RPL26 will inhibit the mTOR pathway, aggravate autophagy and trigger apoptosis.     

The synthesis of ribosomal protein and ribosomal RNA in a rat-mouse hybrid cell line

A rat-mouse hybrid cell line was examined for the presence of ribosomal RNA and ribosomal proteins from both parents. As demonstrated by banding of centromeric heterochromatin, the hybrid cell line contained most of the mouse genome and at least 13 rat chromosomes. The ability of rat, but not mouse, ribosomes to dimerize was used to show that the hybrid contained both rat and mouse 28S ribosomal RNA. Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins indicated the presence of both rat and mouse ribosomal proteins.     

The structure of the gene for ribosomal protein L5 in the archaebacterium Sulfolobus acidocaldarius.

The gene for the ribosomal protein L5 from the archaebacterium Sulfolobus acidocaldarius has been isolated and sequenced. The gene codes for a basic protein of molecular weight 29 165 Da. This protein shows substantial similarity to the equivalent protein from other archaebacteria as well as from yeast, and considerably less similarity to the equivalent eubacterial protein. These results support the concept of the archaebacteria as a monophyletic kingdom more closely related to eukaryotes than to eubacteria.     

Mapping the growth of fungal hyphae: orthogonal cell wall expansion during tip growth and the role of turgor

By computer-enhanced videomicroscopy, we mapped the trajectory of external and internal cell surface markers in growing fungal hyphae to determine the pattern of cell wall expansion during apical growth. Carbon particles (India ink) were chosen as external markers for tip expansion of Rhizoctonia solani hyphae. Irregularities in the growing apical walls of R. solani served as internal markers. Marker movement was traced in captured frames from the videotaped sequences. External and internal markers both followed orthogonal trajectories; i.e., they moved perpendicular to the cell surface regardless of their initial position in the hyphal apex. We found no evidence that the tip rotates during elongation. The discovery that the cell wall of a growing hypha expands orthogonally has major repercussions on two fronts: 1) It supports the long-held view that turgor pressure is the main force driving cell wall expansion. 2) It provides crucial information to complete the mathematical derivation of a three-dimensional model of hyphal morphogenesis based on the vesicle supply center concept. In three dimensions, the vesicle gradient generated by the vesicle supply center is insufficient to explain shape; it is also necessary to know the manner in which the existing surface is displaced during wall expansion.