John G.G. Schoenmakers

Cloning and expression of the gene coding for the transmission blocking target antigen Pfs48/45 of Plasmodium falciparum

The gene encoding the gametocyte/gamete-specific membrane protein Pfs48/45 of Plasmodium falciparum has been cloned. The Pfs48/45 gene is a non-interrupted, single copy gene that codes for a hydrophobic, non-repetitive protein of 448 amino acid residues containing a putative signal peptide at the N-terminus, a hydrophobic C-terminus and 7 potential N-glycosylation sites. Antibodies directed against a Pfs48/45-glutathione-S-transferase fusion protein reacted with both the 45-kDa and 48-kDa proteins of gametocytes. When Pfs48/45 is expressed in the baculovirus-insect cell system the recombinant Pfs48/45 protein is targeted and exposed to the insect cell surface in such a configuration that it is recognized by transmission-blocking anti-45/48-kDa monoclonal antibodies.

Nucleotide sequence and genetic organization of the genome of the N-specific filamentous bacteriophage IKe: Comparison with the genome of the F-specific filamentous phages M13, fd and f1

The nucleotide sequence and genetic organization of the genome of the N-specific filamentous single-stranded DNA phage IKe has been established and compared with that of the F-specific filamentous phages M13, fd and f1 (Ff). The IKe DNA sequence comprises 6883 nucleotides, which is 476 (475) nucleotides more than the nucleotide sequence of the Ff genome. The data indicate that IKe and Ff have evolved from a common ancestor (overall homology approx. 55%) and that their genomes contain ten homologous genes, the order of which is identical. Similar to Ff, the major coat protein and the gene III-encoded pilot protein of IKe are synthesized via precursor molecules. The extent of homology between the genes of IKe and Ff differs significantly from one gene to another. Genes that code for viral capsid proteins are less homologous than genes whose products are involved in the processes of DNA replication and phage morphogenesis. During evolution, large nucleotide sequence rearrangements have occurred in the gene (gene III) whose product is needed for the attachment of the virion to the conjugative pili of the host cell, suggesting that these rearrangements have led to phages with different host specificities. Extensive nucleotide sequence homology was noted between the structural elements involved in DNA replication and phage morphogenesis, indicating that the mechanisms involved in DNA replication and morphogenesis are highly conserved.

Human γ-crystallin genes: A gene family on its way to extinction☆

Abstract During hominoid evolution the γ-crystallins of the lens have decreased in quantity as well as complexity, a change correlated with an increased water content of the lens. To trace the molecular basis for the decrease in γ-crystallin gene expression, we have characterized the structure and expression of the human γ-crystallin gene family. We show that the human γ-crystallin gene family consists of six complete genes (γA, γB, γC, γD, ψγE and ψγF) and one second exon fragment, the γG gene. Model experiments showed that, although the γG sequence is bordered by consensus splice sites, it is most likely transcriptionally inactive in the lens. In the human embryonic lens the γC and γD genes accounted for 81 % of the γ-crystallin transcripts, the γA gene contributed 14% and the γB gene only 5%. The composition of the γ-crystallin mRNA pool changed only after birth, with the γD transcript as the only detectable transcript at ten years of age. The relative activities of the γA, γC and γD promoters in a transient expression system were in agreement with the ratio of their in vivo RNA levels, suggesting that the difference in accumulation of these transcripts is due to differences in the rate of transcription. The γB promoter was much more active than expected and had lost its tissue-specificity. Model experiments showed that the low yield of the γB transcript is due to post-transcriptional processes, most likely RNA instability mediated by third exon sequences. Together with previous data, our results show that the decrease in expression of the γ-crystallin genes in the human lens is the consequence of gene loss (γG), inactivation of coding sequences (ψγE and ψγF), decrease in rate of transcription (γA), increase in rate of RNA turn-over (γB) and a delay in the onset of transcription during development.

Hageman factor, a novel sialoglycoprotein with esterase activity

A novel esterase has been isolated from bovine plasma in a highly purified form. According to electrophoresis and ultracentrifugal sedimentation patterns it represents a homogeneous 7.1 S sialoglycoprotein being composed of 5.9% neutral carbohydrates, 4.8% aminosugars and 4.4% sialic acid. In catalytic amounts it normalizes the prolonged clotting time of plasma deficient in Hageman factor and exhibits a strong esterase activity towards N-benzoyl-l-arginine ethyl ester.

Concerted and divergent evolution within the rat γ-crystallin gene family☆

Abstract The nucleotide sequences of six rat γ-crystallin genes have been determined. All genes have the same mosaic structure: the first exons contain a relatively short (25 to 44 base-pair) 5′ non-coding region and the first nine base-pairs of the coding sequence, the second exons encode protein motifs I and II, while protein motifs III and IV are encoded by the third exons. The third exons also contain a 60 to 67-base-pair long 3′ non-coding region. In the γ1–2 gene, the splice acceptor site of the third exon has been shifted three base-pairs upstream. Hence, the protein product of this gene is one amino acid residue longer. The first introns, though varying in length from 85 to 100 base-pairs, are conserved in sequence. The second introns vary considerably in length (0.9 × 10 3 to 1.9 × 10 3 base-pairs) and sequence. The second exons of the genes show concerted evolution and have undergone multiple gene conversions. In contrast, the third exons show divergent evolution. From the sequences of the third exons, an evolutionary tree of the gene family was constructed. This tree suggests that three of the present genes derive directly from the genes that originated from a tandem duplication of a two-gene cluster. Two duplications of the last gene of the four-gene cluster then yielded the other three genes. Region a′ of the third exon, encoding protein motif III, is variable, while the region encoding protein motif IV (b′) is constant. We postulate that this variability in region a′ is due to a period of radiation after each gene duplication. A comparison of the rat sequences with those of orthologous sequences from other species shows that the variation in region a′ is now preserved. Hence, it might specify the specific functional property of each γ-crystallin protein within the lens.

A novel protein antigen of the malaria parasite Plasmodium falciparum, located on the surface of gametes and sporozoites

A Plasmodium falciparum cDNA clone was isolated of which the insert is transcribed at high rates as a 1.4-kb mRNA in the sexual stages of the malaria parasite. The cDNA clone contains a copy of a non-interrupted gene which codes for a protein of 157 amino acids (Mr = 16607). This 16-kDa protein does not contain repetitive sequences and is characterised by a putative N-terminal signal sequence, a hydrophobic membrane anchor sequence and a highly hydrophilic C-terminal region suggesting that it is an integral membrane protein. Rabbit antisera raised against a synthetic peptide covering amino acids 31-47 of the 16-kDa protein and against recombinant fusion proteins recognised the 16-kDa antigen in protein extracts of gametocytes, macrogamete/zygotes and sporozoites by Western blot analysis. The rabbit antisera also reacted with gametes, gametocytes and sporozoites in a standard immunofluorescence assay. By immunoelectron microscopy using the protein A-gold method the 16-kDa protein could be clearly visualised on the surface of macrogametes and sporozoites, whereas the antigen was not detectable in the asexual erythrocytic stages of the parasite. The 16-kDa antigen of P. falciparum therefore might have the potential to elicit a dual protective immune response against the sporozoite and sexual stage parasites.

Nucleotide sequence and deduced amino acid sequence of a Plasmodium falciparum actin gene

The nucleotide sequence of a Plasmodium falciparum actin gene has been established. The gene codes for a protein of 376 amino acids and is not interrupted by introns. The nucleotide sequence reveals an extreme bias in codon usage. Not less than 85% of the codons possess an A or T at the third position. As has been found for the actins in other unicellular eukaryotes, P. falciparum actin is related both to vertebrate cytoplasmic and vertebrate muscle specific actins. However, the malarial actin is one of the most alpha-like actins hitherto found in lower eukaryotes.

Developmental expression of crystallin genes: In situ hybridization reveals a differential localization of specific mRNAs☆

The time and place of the accumulation of alpha A-, beta B1- and gamma-crystallin RNA in the developing rat lens have been studied by in situ hybridization. alpha A- and gamma-crystallin RNA were first detected in the lens vesicle, while beta B1-crystallin RNA could be seen only after elongation of the primary fiber cells. Both beta B1- and gamma-crystallin RNA were confined to the fiber cells of fetal lenses, while alpha A-crystallin mRNA could also be detected in the epithelial cells. A quantification of the hybridization pattern obtained in the differentiation zone of the newborn rat lens showed that alpha A-crystallin RNA is concentrated in the cortical zone. alpha B-crystallin mRNA has the same distribution pattern. beta B1-crystallin RNA was relatively poorly detectable by in situ hybridization in both fetal and newborn rat lenses. The grain densities obtained with this probe increased from the periphery of the lens toward the interior, indicating that beta B1-crystallin RNA accumulated during differentiation of the secondary fiber cells. A similar accumulation pattern was obtained for gamma-crystallin mRNA, but, unexpectedly, this RNA could also be detected in the elongating epithelial cells. Our results show that gamma-crystallin RNA starts to accumulate as soon as visible elongation of epithelial cells occurs, during differentiation of the primary as well as the secondary fiber cells.

Rise and fall of crystallin gene messenger levels during fibroblast growth factor induced terminal differentiation of lens cells.

Explanted rat lens epithelial cells differentiate synchronously in vitro to lens fiber cells in the presence of basic fibroblast growth factor (bFGF). We have monitored the expression of the three rat crystallin gene families, the alpha-, beta-, and gamma-crystallin genes, during this process. The expression of these gene families is sequentially activated, first the alpha-crystallin genes at Day 1, then the beta-crystallin genes at Day 3, and finally the gamma-crystallin genes at Day 8. The steady state levels of alpha- and beta-crystallin mRNA are not affected by incubation with actinomycin D, suggesting that these mRNAs are stable. Nevertheless, all crystallin mRNAs disappear from the differentiated explants between Days 10 and 11, a process signaled by bFGF. At this time a novel abundant mRNA appears. Cloning and sequencing showed that this mRNA encoded aldose reductase. Our results suggest a novel model for the regulation of crystallin synthesis during lens cell differentiation: a gene pulse delivers a certain amount of stable mRNA, this mRNA is removed at a later stage of differentiation by a stage-specific breakdown mechanism. Each of these regulatory steps requires a signal from bFGF.

Further studies on the ribonuclease inhibitor from rat liver: stability and other properties.

Abstract A number of properties including stability of the ribonuclease inhibitor from rat liver have been described. The enzyme-inhibitor complex is formed instantaneously and is strongly dependent on pH and Mg 2+ concentration. The pH optimum of the inhibitor coincides with the optimal pH value of pancreatic ribonuclease. Proteolytic enzymes destroy the inhibitory activity, whereas dithiothreitol stabilizes the purified inhibitor at neutral or slightly alkaline pH. At acid pH the factor is rather stable.