PRB2/1 fusion gene: a product of unequal and homologous crossing-over between proline-rich protein (PRP) genes PRB1 and PRB2.

The PRB2/1 fusion gene is produced by homologous and unequal crossing-over between PRB1 and PRB2 genes that code for basic salivary proline-rich proteins (PRPs). To determine the molecular basis for the PRB2/1 fusion gene, the DNA sequence was determined for the PRB2/1 gene and was compared with those of the PRB1 and PRB2 genes. From these comparisons, the crossing-over is postulated to occur in a 743-bp region of identity, with only 1-bp mismatch between the PRB1 and PRB2 genes, in the third intron outside the coding region of the two genes. This region of virtual complete identity is the largest found between any of the six closely linked PRB genes and may facilitate recombination. Since the coding region of PRB1 is completely absent from the PRB2/1 gene, salivas from two white PRB2/1 homozygotes were studied to determine which polymorphic PRPs were missing from the salivas. Polymorphic PRPs Pe, PmF, PmS, and Ps were found to be missing from the salivas. However, a white individual lacking the same salivary PRPs is a PRB2/1 heterozygote with one PRB1 allele. The explanation for the missing salivary proteins in this individual is unknown. The PRB2/1 gene is relatively frequent in several populations of unrelated individuals, including American blacks (n = 41), American Utah whites (n = 76), and mainland Chinese (n = 131), with gene frequencies of .22, .06, and .09, respectively. Evidence for the occurrence of PRB1/2 heterozygotes is also presented.     

PRB1, PRB2, and PRB4 coded polymorphisms among human salivary concanavalin-A binding, II-1, and Po proline-rich proteins.

Six closely linked PRP (proline-rich protein) genes code for many salivary PRPs that show frequent length and null variants. From determined protein sequences and DNA sequence analysis of variant alleles, we here report the coding and molecular basis for Con (concanavalin A-binding) and Po (parotid "o") protein polymorphisms. The Con1 glycoprotein is encoded in exon 3 of a PRB2 allele (PRB2L CON1+) with a potential N-linked glycosylation site. Because of a probable gene conversion encompassing > or = 684 bp of DNA, the "PRB2-like" Con2 glycoprotein is encoded in exon 3 of a PRB1 allele (PRB1M CON2+) with a potential glycosylation site. The PmF protein is also encoded in the PRB1M CON2+ allele, thus explaining the previously reported association between Con2 and PmF proteins. A PRB2L CON1 allele contains a single nt missense change [TCT(Ser)-->CCT (Pro)] that abolishes the potential N-linked glycosylation site (NKS-->NKP) in the Con1 protein, and this explains the Con- type. The Po protein and a glycoprotein (II-1) are encoded in the PRB4 gene, and both proteins are absent in the presence of a mutation in the PRB4M PO- allele that contains a single nt change (G--C) at the +1 invariant position of the intron 3 5'donor splice site. The genetically determined absence of the II-1 glycoprotein leads to altered in vitro binding of Streptococcus sanguis 10556 to salivary proteins, which suggests a biological consequence for null mutations of the PRB4 gene.     

Genetic polymorphisms of Pe and Po salivary proteins with probable linkage of their genes to the salivary protein gene complex (SPC)

Two new genetic polymorphisms (Pe and Po) are found in human parotid saliva. Each polymorphism is determined by the autosomal inheritance of one expressed (dominant) and one unexpressed (recessive) allele. Autosomal inheritance is supported by studies of 63 families including 264 children for Pe and 57 families including 242 children for Po. For randomly collected salivas, gene frequencies in 317 whites are Pe+ = 0.76 and Pe- = 0.24; in 408 whites, Po+ = 0.75 and Po- = 0.25; in 51 blacks, Pe+ = 0.76 and Pe- = 0.24; and in 59 blacks, Po+ = 0.77 and Po- = 0.23. Both Pe and Po proteins react immunologically with polyclonal antisera prepared to proline-rich proteins PRPs. The Pe protein has an isoelectric point of approximately pH 6.1-6.3, and the Po protein has an isoelectric point greater than pH 8.0. In randomly collected salivas, the Pe and Po proteins are associated with other known salivary PRPs. The Pe protein is most strongly associated with the CON 1 and Ps proteins, is less strongly associated with the Pr and Pa proteins, and is not significantly associated with the PmF, PmS, PIF, Db, Con 2, or Gl proteins. If it is assumed that the strength of these associations (presumed linkage disequilibrium) may be related in part to map distance, then these data roughly fit the linear order of PRP genes as previously determined from recombination data derived from family linkage studies. The Po protein is associated with the PmS protein. There is evidence for probable linkage of Pe and Po to the SPC (salivary protein gene complex): Pe to Pa (nine families, lod score at theta = 0 is 2.67) and Po to CON 2 (three families, lod score at theta = 0 is 2.35).     

Inheritance of the human salivary proline-rich proteins: A reinterpretation in terms of six loci forming two subfamilies

DNA studies suggest that six loci control the synthesis of human salivary proline-rich proteins (PRPs). Genes at two of these loci (proposed names, PRH1 and PRH2) contain regions that strongly hybridize to a probe made from a cDNA in which sites for the restriction enzyme HaeIII occur repeatedly; they code for the acidic PRPs. Genes at the remaining four loci (PRB1, PRB2, PRB3, and PRB4) contain regions that strongly hybridize to a probe with repeated BstN1 sites; they probably code for the basic and glycosylated PRPs. In contrast to these data suggesting six loci forming two gene subfamilies, studies of protein polymorphisms and families have led to the postulation of 13 loci with 11 common null alleles. The discrepancy in the number of loci is partly resolved by the hypothesis that the three acidic PRPs, Db, Pa, and PIF, are coded for by alleles at one of the HaeIII-type loci rather than by three discrete loci.This work was supported by National Institutes of Health Grants DEO 3658-19 (E.A.) and GM 20069 (O.S.). This is paper No. 2774 from the Laboratory of Genetics, University of Wisconsin—Madison.     

Alleles at the PRB3 locus coding for a disulfide-bonded human salivary proline-rich glycoprotein (Gl 8) and a null in an Ashkenazi Jew.

From electrophoretic analysis, we identified in the saliva of an Ashkenazi Jew a disulfide-bonded major glycoprotein variant (Gl 8) that is a product of the proline-rich protein (PRP) locus PRB3. A previous study of this variant protein misidentified it as Pa 2 and as a product of a different PRP locus. The other PRB3 allele in this individual is an apparent null. To identify the mutations, we sequenced the tandemly repetitious exon 3 (the major protein-coding portions) of both alleles. A CGT----TGT (Arg----Cys) mutation was found in one allele (PRB3Scys), which accounts for the disulfide-bonded and peroxidase-modifying properties of Gl 8. A single nucleotide insertion was found in the other allele (PRB3Mnull) that leads to a frameshift with a premature termination codon that causes an apparent lack of gene expression. Null alleles are frequent at PRP loci coding for basic and glycosylated PRPs, and the mechanism described might explain other null phenotypes among PRPs. From nucleotide comparisons, a model of intragenic unequal crossing-over is proposed to explain, in part, the generation of the PRB3Mnull allele. The Gl 8 protein variant is found in Ashkenazi Jews (gene frequency around .008) but not in the general white, black, or Japanese populations. It is interesting that products of different PRP genes, Gl 8 from PRB3 and Pa 1 from PRH1, are both disulfide bonded and probably modify salivary peroxidase (part of an important intraoral antibacterial system) through formation of disulfide-bonded heterodimers.     

Versatile roles of Arabidopsis plastid ribosomal proteins in plant growth and development

A lack of individual plastid ribosomal proteins (PRPs) can have diverse phenotypic effects in Arabidopsis thaliana, ranging from embryo lethality to compromised vitality, with the latter being associated with photosynthetic lesions and decreases in the expression of plastid proteins. In this study, reverse genetics was employed to study the function of eight PRPs, five of which (PRPS1, ‐S20, ‐L27, ‐L28 and ‐L35) have not been functionally characterised before. In the case of PRPS17, only leaky alleles or RNA interference lines had been analysed previously. PRPL1 and PRPL4 have been described as essential for embryo development, but their mutant phenotypes are analysed in detail here. We found that PRPS20, ‐L1, ‐L4, ‐L27 and ‐L35 are required for basal ribosome activity, which becomes crucial at the globular stage and during the transition from the globular to the heart stage of embryogenesis. Thus, lack of any of these PRPs leads to alterations in cell division patterns, and embryo development ceases prior to the heart stage. PRPL28 is essential at the latest stages of embryo–seedling development, during the greening process. PRPS1, ‐S17 and ‐L24 appear not to be required for basal ribosome activity and the organism can complete its entire life cycle in their absence. Interestingly, despite the prokaryotic origin of plastids, the significance of individual PRPs for plant development cannot be predicted from the relative phenotypic severity of the corresponding mutants in prokaryotic systems.     

Digenic junctional epidermolysis bullosa: mutations in COL17A1 and LAMB3 genes

Junctional epidermolysis bullosa (JEB), a genetically heterogeneous group of blistering skin diseases, can be caused by mutations in the genes encoding laminin 5 or collagen XVII, which are components of the hemidesmosome-anchoring filament complex in the skin. Here, a family with severe nonlethal JEB and with mutations in genes for both proteins was identified. The index patient was compound heterozygous for the COL17A1 mutations L855X and R1226X and was heterozygous for the LAMB3 mutation R635X. As a consequence, two functionally related proteins were affected. Absence of collagen XVII and attenuated laminin 5 expression resulted in rudimentary hemidesmosome structure and separation of the epidermis from the basement membrane, with severe skin blistering as the clinical manifestation. In contrast, single heterozygotes carrying either (1) one or the other of the COL17A1 null alleles or (2) a double heterozygote for a COL17A1 and a LAMB3 null allele did not have a pathological skin phenotype. These observations indicate that the known allelic heterogeneity in JEB is further complicated by interactions between unlinked mutations. They also demonstrate that identification of one mutation in one gene is not sufficient for determination of the genetic basis of JEB in a given family.     

Binding of Escherichia coli ribosomal proteins to 23S RNA under reconstitution conditions for the 50S subunit.

The RNA binding capacity of 50S proteins from E. coli ribosomes has been tested under improved conditions; purified proteins active in reconstitution assays were used, and the binding was studied under the conditions of the total reconstitution procedure for the 50S subunit. The results are: 1) Interaction of 23S RNA was found with 17 proteins, namely L1, L2, L3, L4, L7/L12, L9, L10, L11, L15, L16, L17, L18, L20, L22, L23, L24 and L29. 2) The proteins L1, L2, L3, L4, L9, L23 and L24 bound to 23S RNA at a level of about one copy per RNA molecule, whereas L20 could bind more than one copy (no saturation was observed at 1.8 copies per 23S RNA), and the other proteins bound 0.2--0.6 copies per RNA. 3) L1, L3, L7/L12 showed a slight binding to 16S RNA, L26 (identical with S20) strong binding to 16S RNA. 4) The binding of L2, L7/L12, L10, L11, L15, L16 and L18 was preparation sensitive, i.e. the binding ability changed notably from preparation to preparation. 5) All proteins bound equally well to 23S RNA in presence of 4 and 20 mM Mg2+, respectively, except L2, L3, L4, L7/L12, L9, L10, L15, L16 and L18, which bound less strongly at 20 mM than at 4 mM Mg2+.     

Length Polymorphisms in Human Proline-Rich Protein Genes Generated by Intragenic Unequal Crossing over

Southern blot hybridization analysis of genomic DNAs from 44 unrelated individuals revealed extensive insertion/deletion polymorphisms within the BstNI-type loci (PRB1, PRB2, PRB3 and PRB4) of the human proline-rich protein (PRP) multigene family. Ten length variants were cloned, including alleles at each of the four PRB loci, and in every case the region of length difference was localized to the tandemly repetitious third exon. DNA sequences covering the region of length variation were determined for seven of the alleles. The data indicate (1) that the PRB loci can be divided into two subtypes, PRB1 plus PRB2, and PRB3 plus PRB4, and (2) that the length differences result from different numbers of tandem repeats in the third exons. Variant chromosomes were also identified with different numbers of PRP loci resulting from homologous but unequal exchange between the PRB1 and PRB2 loci. The overall data are compatible with the observed length variants having been generated via homologous but unequal intragenic exchange. The results also indicate that these crossover events are sensitive to the amount of homology shared between the interacting DNA strands. Allelic length variants have arisen independently at least 20 times at the PRB loci, but only one has been detected at a PRH locus. Comparison of the detailed structures of the repetitious regions in PRB and PRH loci shows that the repeats in PRB genes are very similar to each other in sequence and in length. The PRH genes contain fewer repeats, which differ considerably in their individual lengths. These differences suggest that the larger number of length variants in PRB genes is related to their greater ease of homologous but unequal pairing compared to PRH genes.     

SPO11.2 is essential for programmed double‐strand break formation during meiosis in bread wheat (Triticum aestivum L.)

Meiotic recombination is initiated by formation of DNA double‐strand breaks (DSBs). This involves a protein complex that includes in plants the two similar proteins, SPO11‐1 and SPO11‐2. We analysed the sequences of SPO11‐2 in hexaploid bread wheat (Triticum aestivum), as well as in its diploid and tetraploid progenitors. We investigated its role during meiosis using single, double and triple mutants. The three homoeologous SPO11‐2 copies of hexaploid wheat exhibit high nucleotide and amino acid similarities with those of the diploids, tetraploids and Arabidopsis. Interestingly, however, two nucleotides deleted in exon‐2 of the A copy lead to a premature stop codon and suggest that it encodes a non‐functional protein. Remarkably, the mutation was absent from the diploid A‐relative Triticum urartu, but present in the tetraploid Triticum dicoccoides and in different wheat cultivars indicating that the mutation occurred after the first polyploidy event and has since been conserved. We further show that triple mutants with all three copies (A, B, D) inactivated are sterile. Cytological analyses of these mutants show synapsis defects, accompanied by severe reductions in bivalent formation and numbers of DMC1 foci, thus confirming the essential role of TaSPO11‐2 in meiotic recombination in wheat. In accordance with its 2‐nucleotide deletion in exon‐2, double mutants for which only the A copy remained are also sterile. Notwithstanding, some DMC1 foci remain visible in this mutant, suggesting a residual activity of the A copy, albeit not sufficient to restore fertility.     

Neutral and DEAE dextrans as tracers for assessing lung microvascular barrier permeability and integrity.

Steady-state lymph-to-plasma concentration ratios (L/Ps) of neutral dextrans, cationic DEAE dextrans, and endogenous proteins were determined under normal and increased permeability conditions in six unanesthetized yearling sheep prepared with chronic lung lymph fistulas. Fluorescent dextrans with radii ranging from 1 to 30 nm were intravenously infused, and after 24 h, perilla ketone (PK) was given to alter permeability while the dextran infusion was maintained. Plasma and lymph samples were collected before and after PK administration and analyzed for dextran and protein concentrations after high-performance liquid chromatography size separation. Under both baseline and increased permeability conditions, DEAE dextrans had higher L/Ps than neutral dextrans of similar size but lower L/Ps than proteins of similar size. Comparison of L/Ps before and after PK revealed that the percentage change in permeability for neutral and DEAE dextrans was significantly larger than that for proteins. These results suggest that 1) the pulmonary microvascular barrier behaves as a net negative barrier, 2) some transport mechanisms for proteins and dextrans are different, and 3) neutral and cationic dextrans are more sensitive markers than proteins of the same size for assessing changes in pulmonary capillary permeability.     

Development of Transgenic Mice Containing an Introduced Stop Codon on the Human Methylmalonyl-CoA Mutase Locus

The mutation R403stop was found in an individual with mut⁰ methylmalonic aciduria (MMA) which resulted from a single base change of C→T in exon 6 of the methylmalonyl-CoA mutase gene (producing a TGA stop codon). In order to accurately model the human MMA disorder we introduced this mutation onto the human methylmalonyl-CoA mutase locus of a bacterial artificial chromosome. A mouse model was developed using this construct.The transgene was found to be intact in the mouse model, with 7 copies integrated at a single site in chromosome 3. The phenotype of the hemizygous mouse was unchanged until crossed against a methylmalonyl-CoA mutase knockout mouse. Pups with no endogenous mouse methylmalonyl-CoA mutase and one copy of the transgene became ill and died within 24 hours. This severe phenotype could be partially rescued by the addition of a transgene carrying two copies of the normal human methylmalonyl-CoA mutase locus. The “humanized” mice were smaller than control litter mates and had high levels of methylmalonic acid in their blood and tissues.This new transgenic MMA stop codon model mimics (at both the phenotypic and genotypic levels) the key features of the human MMA disorder. It will allow the trialing of pharmacological and, cell and gene therapies for the treatment of MMA and other human metabolic disorders caused by stop codon mutations.     

The structure and evolution of the human salivary proline-rich protein gene family

We present the nucleotide sequences of four members of the six-member human salivary prolinerich protein (PRP) gene family. The four genes are PRB1 and PRB2, which encode basic PRPs, and PRB3 and PRB4, which encode glycosylated PRPs. Each PRB gene is approximately 4.0 kb in length and contains four exons, the third of which is entirely composed of 63-bp tandem repeats and encodes the proline-rich portion of the protein products. Exon 3 contains different numbers of tandem repeats in the different PRB genes. Variation in the numbers of these repeats is also responsible for length variations in different alleles of the PRB genes. We have determined a probable evolutionary history of the human PRP gene family by comparing the nucleotide sequences of the six PRP genes. The present-day six PRP loci probably evolved from a single ancestral gene by four sequential gene duplications, leading to six genes that fall into three subsets, each consisting of two genes. During this evolutionary process, multiple rearrangements and gene conversion occurred mainly in the region from the 3 end of IVS2 and the 3 end of exon 3.     

Emphysema associated with complete absence of alpha 1- antitrypsin in serum and the homozygous inheritance [corrected] of a stop codon in an alpha 1-antitrypsin-coding exon.

Homozygous inheritance of the null bellingham alpha 1-antitrypsin (alpha 1AT) gene is associated with early-onset emphysema, resulting from the lack of alpha 1AT to protect the lung from neutrophil elastase. Cloning and sequencing of the null bellingham gene demonstrated that the promoter region, coding exons, and all exon-intron junctions were normal except for a single base substitution in exon III, causing the normal lys217 (AAG) to become a stop codon (TAG). Evaluation of genomic DNA of family members by using oligonucleotides directed toward this region demonstrated that the index case had inherited this mutation in a homozygous fashion. Although the consequences to the individual (i.e., emphysema) are identical to those associated with the common homozygous Z mutation, the homozygous null bellingham form of alpha 1AT deficiency has a very different genetic basis.     

Description of a genetic polymorphism of a human proline-rich salivary protein,Pc, and its relationship to other proteins in the salivary protein complex (SPC)

A new polymorphism, Pc, has been identified in human saliva. Two proteins, Pc 1 and Pc 2, are determined by alleles Pc1 and Pc2, respectively, which show autosomal codominant inheritance. No null phenotype has been encountered in 225 randomly collected salivas. The frequencies of the two alleles differ in the Black and White American populations, with Pc1 and Pc2 being 0.670 and 0.330 in the Black (N = 47) and 0.461 and 0.539 in the White (N = 178) populations, respectively. The alleles are in equilibrium in the two populations and segregation analyses (30 families) do not suggest the existence of a null allele in either population. Of seven polymorphic human salivary proteins determined by genes in the salivary protein complex (SPC), Pc phenotypes show association only with Ps phenotypes. Based on that association, our linkage studies, and the biochemical similarities with other SPC proteins, we tentatively conclude that Pc is a member of the SPC, bringing the total number of genes in that group to 13.