cDNA cloning, expression pattern, genomic structure and chromosomal location of RAB6KIFL, a human kinesin-like gene

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal 35 kDa, plant 55 kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.     

Functional analysis of proteins involved in Plasmodium falciparum merozoite invasion of red blood cells

Plasmodium falciparum causes the most lethal form of malaria in humans and is responsible for over two million deaths per year. The development of a vaccine against this parasite is an urgent priority and potential protein targets include those on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to transfect P. falciparum has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. In this review, we describe the use of this technology to examine the role of merozoite antigens in erythrocyte invasion and to address their potential as vaccine candidates.     

Apical membrane antigen 1 plays a central role in erythrocyte invasion by Plasmodium species

Apical membrane antigen 1 (AMA1) is an asexual blood-stage protein expressed in the invasive merozoite form of Plasmodia species, which are the causative agent of malaria. We have complemented the function of Plasmodium falciparum AMA1 (PfAMA1) with a divergent AMA1 transgene from Plasmodium chabaudi (PcAMA1). It was not possible to disrupt the PfAMA1 gene using 'knock-out' plasmids, although we demonstrate that the PfAMA1 gene can be targeted by homologous recombination. These experiments suggest that PfAMA1 is critical, perhaps essential, for blood-stage growth. Importantly, we showed that PcAMA1 expression in P. falciparum provides trans-species complementation to at least 35% of the function of endogenous PfAMA1 in human red cells. Furthermore, expression of this transgene in P. falciparum leads to more efficient invasion of murine erythrocytes. These results indicate an important role for AMA1 in the invasion of red blood cells (RBCs) across divergent Plasmodium species.     

Revealing the molecular determinants of gender in malaria parasites

Malaria parasites have a complex life cycle with asexual multiplication in a vertebrate host and obligate sexual reproduction in the mosquito; however, commitment to sexual development begins in the vertebrate with differentiation of female and male gametocytes. In this issue of Cell, Khan et al. (2005) used elegant approaches to purify male and female gametocytes and elucidated their respective proteome, providing the basis for understanding sexual development in this pathogen.     

ERK8, a new member of the mitogen-activated protein kinase family

The ERKs are a subfamily of the MAPKs that have been implicated in cell growth and differentiation. By using the rat ERK7 cDNA to screen a human multiple tissue cDNA library, we identified a new member of the ERK family, ERK8, that shares 69% amino acid sequence identity with ERK7. Northern analysis demonstrates that ERK8 is present in a number of tissues with maximal expression in the lung and kidney. Fluorescence in situ hybridization localized the ERK8 gene to chromosome 8, band q24.3. Expression of ERK8 in COS cells and bacteria indicates that, in contrast to constitutively active ERK7, ERK8 has minimal basal kinase activity and a unique substrate profile. ERK8, which contains two SH3-binding motifs in its C-terminal region, associates with the c-Src SH3 domain in vitro and co-immunoprecipitates with c-Src in vivo. Co-transfection with either v-Src or a constitutively active c-Src increases ERK8 activation indicating that ERK8 can be activated downstream of c-Src. ERK8 is also activated following serum stimulation, and the extent of this activation is reduced by pretreatment with the specific Src family inhibitor PP2. The ERK8 activation by serum or Src was not affected by the MEK inhibitor U0126 indicating that activation of ERK8 does not require MEK1, MEK2, or MEK5. Although most closely related to ERK7, the relatively low sequence identity, minimal basal activity, and different substrate profile identify ERK8 as a distinct member of the MAPK family that is activated by an Src-dependent signaling pathway.     

Identification of the substrate-binding sites of 2'-5'-oligoadenylate synthetase

2'-5'-Oligoadenylate synthetases are interferon-induced enzymes that upon activation by double-stranded RNA polymerize ATP to 2'-5'-linked oligoadenylates. In our continuing effort to understand the mechanism of catalysis by these enzymes, we used photo affinity cross-linking and peptide mapping to identify the substrate-binding sites of the P69 isozyme of human 2'-5'-oligoadenylate synthetases. Radiolabeled azido 2'-5'-oligoadenylate dimers were enzymatically synthesized and used as ligands for cross-linking to the P69 protein by exposure to ultraviolet light. The radiolabeled protein was digested with trypsin, and two ligand-cross-linked peptides were purified by immobilized aluminum affinity chromatography followed by reverse phase high pressure liquid chromatography. The peptides were identified by mass spectrometry and peptide sequencing and were found to correspond to residues 420-425 and 539-547 of P69. To examine the functional importance of the cross-linking sites, specific residues in the two peptides were mutated. When residues in the two sites were mutated individually, ligand cross-linking was selectively eliminated at the mutated site, and the enzyme activity was lost almost completely. Using substrates that can serve either as a donor or as an acceptor but not both, we could identify one of the sites as the acceptor and the other as the donor site.     

Comparative genome analysis of Mycoplasma pneumoniae

Background

Mycoplasma pneumoniae is a common pathogen that causes upper and lower respiratory tract infections in people of all ages, responsible for up to 40 % of community-acquired pneumonias. It also causes a wide array of extrapulmonary infections and autoimmune phenomena. Phylogenetic studies of the organism have been generally restricted to specific genes or regions of the genome, because whole genome sequencing has been completed for only 4 strains. To better understand the physiology and pathogenicity of this important human pathogen, we performed comparative genomic analysis of 15 strains of M. pneumoniae that were isolated between the 1940s to 2009 from respiratory specimens and cerebrospinal fluid originating from the USA, China and England.

Results

Illumina MiSeq whole genome sequencing was performed on the 15 strains and all genome sequences were completed. Results from the comparative genomic analysis indicate that although about 1500 SNP and indel variants exist between type1 and type 2 strains, there is an overall high degree of sequence similarity among the strains (>99 % identical to each other). Within the two subtypes, conservation of most genes, including the CARDS toxin gene and arginine deiminase genes, was observed. The major variation occurs in the P1 and ORF6 genes associated with the adhesin complex. Multiple hsdS genes (encodes S subunit of type I restriction enzyme) with variable tandem repeat copy numbers were found in all 15 genomes.

Conclusions

These data indicate that despite conclusions drawn from 16S rRNA sequences suggesting rapid evolution, the M. pneumoniae genome is extraordinarily stable over time and geographic distance across the globe with a striking lack of evidence of horizontal gene transfer.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1801-0) contains supplementary material, which is available to authorized users.