Partial DNA sequencing of Douglas-fir cDNAs used for RFLP mapping

 DNA sequences from 87 Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) cDNA RFLP probes were determined. Sequences were submitted to the GenBank dbEST database and searched for similarity against nucleotide and protein databases using the BLASTn and BLASTx programs. Twenty-one sequences (24%) were assigned putative functions; 18 of which were from plant species. Six sequences aligned with conifer genes, including genes from Douglas-fir. Similarities among the 87 sequences were revealed by analyses with FASTA, suggesting either redundancy or isoforms of the same gene. Assignment of putative functions to anonymous cDNA mapped markers will increase the understanding of structural gene organization of the Douglas-fir genome. Received: 10 April 1998 / Accepted: 29 April 1998     

Codominant PCR-based markers for Pinus taeda developed from mapped cDNA clones

 We report a strategy for developing codominant PCR-based genetic markers by using sequenced cDNA clones from loblolly pine (Pinus taeda L.). These clones were previously used as probes for detecting restriction fragment length polymorphisms (RFLPs) to generate linkage maps. After assessing the complexity of banding patterns from Southern blots, we selected clones representing relatively simple gene families, and then determined nucleotide sequences for about 200 bp at each end of the cDNA inserts. Specific PCR primers were designed to amplify samples of genomic DNA derived from two loblolly pine mapping populations. Polymorphisms were detected after digesting the amplified DNA fragments with a battery of restriction endonucleases, and most polymorphisms were inherited in a Mendelian fashion. These newly identified genetic markers are codominant and relatively simple to use. By assaying DNA from individuals used to construct RFLP maps, we show that most of these markers map to the same position as the RFLP loci detected using their corresponding cDNAs as probes, implying that these markers have been converted from RFLP to PCR-based methods. These PCR-based markers will be useful for genome mapping and population genetics. Received: 10 February 1998 / Accepted: 25 February 1998     

Chromosomal assignment of gene sequences coding for protein disulphide isomerase (PDI) in wheat

 Three different probes, obtained by PCR amplification and labelling of different segments of a PDI cDNA clone from common wheat, were used to identify and assign to wheat chromosomes the gene sequences coding for protein disulphide isomerase (PDI). One of these probes, containing the whole coding region except for a short segment coding for the C-terminal sequence, displayed defined and specific RFLP patterns. PDI gene sequences were consequently assigned to wheat chromosome arms 4BS, 4DS, 4AL and 1BS by Southern hybridisation of EcoRI- HindIII- and BamHI-digested total DNA of nulli-tetrasomic and di-telosomic lines of Chinese Spring. This probe was also employed for assessing the restriction fragment length polymorphism in several hexaploid and tetraploid cultivated wheats. These showed considerable conservation at PDI loci; in fact polymorphism was only observed for the chromosome 1B fragment. Received: 7 July 1998 / Accepted: 14 August 1998     

Chromosomal localization of human aspartate aminotransferase genes by in situ hybridization

Summary The localization of the human genes for cytosolic and mitochondrial aspartate aminotransferase (AspAT) has been determined by chromosomal in situ hybridization with specific human cDNA probes previously characterized in our laboratory. The cytosolic AspAT gene is localized on chromosome 10 at the interface of bands q241–q251. Mitochondrial AspAT is characterized by a multigene family located on chromosomes 12 (p131–p132), 16 (q21), and 1 (p32–p33 and q25–q31). Genomic DNA from ten blood donors was digested by ten restriction enzymes, and Southern blots were hybridized with the two specific probes. Restriction fragment length polymorphism was revealed in only one case for cytosolic AspAT, with PvuII, while no polymorphism for mitochondrial AspAT was found.     

cDNA cloning of luteinizing hormone subunits from brushtail possum and red kangaroo

Luteinizing hormone (LH) plays an important role in the reproductive cycles of all mammals. There is a large amount of both nucleotide and amino acid sequence data available for LH from eutherian mammals, but little is known about the primary structure of LH in marsupials. We have used consensus PCR primers to generate specific probes for screening pituitary cDNA libraries and report the cloning of the cDNAs encoding the α-subunit of LH (also shared by a number of other glycoprotein hormones) and the LH-specific β-subunit, from the common brushtail possum, Trichosurus vulpecula, and the red kangaroo, Macropus rufus. Southern blotting experiments indicated that both genes are probably present as single copies. Comparison of the deduced marsupial protein sequences with homologous sequences from other vertebrates revealed a high degree of conservation, especially for the α-subunit. These sequences represent the first complete primary structures for a marsupial glycoprotein hormone to have been elucidated. Received: 30 January 1998 / Accepted: 10 April 1998     

Cloning and nucleotide sequence of human liver cDNA encoding for cystathionine gamma-lyase.

We have cloned and sequenced a full-length cDNA (1083 bp) encoding the human liver cystathionine-gamma-lyase enzyme (cystathionase). The human cystathionase sequence presented a substantial deletion of 132 bases (44 amino acids) compared to that reported for rat cystathionase, and of 135 bases (45 amino acids) compared to that reported for yeast cystathionase. After re-alignment for the missing nucleotides, the human cDNA sequence shows significant amino acid homology to that for the rat enzyme (85%) and the yeast enzyme (50%). A search for an undeleted cDNA, by the polymerase chain reaction, yielded a second clone which contained the missing 132 bases. Flanking nucleotides in the latter clone were identical to those in the cDNA clone containing the deletion. The two forms of human cystathionase deduced from the two cDNA clones may be derived from two different genes or may be splice variants.     

An elicitor-induced cDNA from aerial yam (Dioscorea bulbifera L.) encodes a pathogenesis-related type 4 protein

We established Dioscorea bulbifera (aerial yam) cell suspension cultures to study the expression of defense-related genes upon elicitation with the yam pathogenic ascomycete Colletotrichum gloeosporioides. The induction of phenylalanine-ammonia lyase (PAL) mRNA, coding for a key enzyme involved in phytoalexin biosynthesis, was observed upon elicitation. Using RT-PCR, we isolated an elicited cDNA clone with an open reading frame of 453 nucleotides which showed high homology to cDNA sequences of pathogenesis-related proteins belonging to the PRP-4 group. Yam PRP-4 expression is increased in elicited cell cultures as well as in elicited leaves, and is encoded by a small multigene family. This is the first example for the cloning of a cDNA that might be involved in defense reactions of yam plants. Received: December 1997 / Revision received: June 1998 / Accepted: November 1998     

Molecular deletion patterns in Duchenne and Becker type muscular dystrophy

Summary DNA from 80 Duchenne (DMD) and 15 Becker (BMD) index patients was analyzed with 12 genomic probes and the total cDNA. Deletions were detected in 24 DMD (30%) and 10 BMD patients (67%) by genomic probes alone, mostly p20, pXJ, and/or pERT87. All deletions were confirmed by cDNA probes, and an additional 29 DMD deletions were detected, resulting in a total of 63/95 deletions (66%). The majority of the deletions are localized between kb 6.7 and 9.7 of the cDNA; a smaller group, between kb 0.5 and 3.5. Of the deletions, 90% are detected by the three cDNA probes 1–2a, 7, and 8. This can be applied to strategies for carrier detection and prenatal diagnosis. The order of 13 exon-containing HindIII fragments in the region between probes 7 and 9–10, where most of the deletions are found, could be defined. The deletion patterns in DMD and BMD patients are different and well in accordance with the “reading frame theory” of Monaco and coworkers. Thus our findings indicate that a DMD or BMD phenotype may be predicted according to the breakpoint position and the number of deleted exons.     

An RFLP linkage map for loblolly pine based on a three-generation outbred pedigree

A genetic linkage map for loblolly pine (Pinus taeda L.) was constructed using segregation data from a three-generation outbred pedigree consisting of four grandparents, two parents, and 95 F₂ progeny. The map was based predominantly on restriction fragment length polymorphism (RFLP) loci detected by cDNA probes. Sixty-five cDNA and three genomic DNA probes revealed 90 RFLP loci. Six polymorphic isozyme loci were also scored. One-fourth (24%) of the cDNA probes detected more than 1 segregating locus, an indication that multigene families are common in pines. As many as six alleles were observed at a single segregating locus among grandparents and it was not unusual for the progeny to segregate for three or four alleles per locus. Multipoint linkage analysis placed 73 RFLP and 2 isozyme loci into 20 linkage groups; the remaining 17 RFLP and 4 isozyme loci were unlinked. The mapped RFLP probes provide a new set of codominant markers for genetic analyses in loblolly pine.     

Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization

Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross-hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.     

Construction of a saturated linkage map for Prunus using an almond×peach F2 progeny

 A map with 246 markers (11 isozymes and 235 RFLPs) was constructed using an interspecific F₂ population between almond (cv Texas) and peach (cv Earlygold). RFLPs were obtained using 213 probes from the genomic and cDNA libraries of different species (almond, peach, P. ferganensis, cherry, plum and apple), including 16 almond probes which correspond to known genes. All markers were distributed in eight linkage groups, the same as the basic chromosome number of the genus, covering a total distance of 491 cM. The average map density was 2.0 cM/marker and only four gaps of 10 cM or more were found; the two largest gaps were 12cM each. This map was compared with one constructed previously with an intraspecific almond population sharing 67 anchor loci. Locus order was nearly identical and distances were not significantly different. A large proportion of the mapped loci (46%) had skewed segregations; in approximately half of them, the distortion was due to an excess of heterozygotes. One of the distorted regions could be associated with the position of the self-incompatibility gene of almond. Received: 6 November 1997 / Accepted: 26 May 1998     

Sheep linkage mapping: restriction fragment length polymorphism detection with heterologous cDNA probes

Summary. A selection of cattle, human and sheep cDNA probes were screened against sheep genomic DNA, cut with 10 different restriction enzymes, to assess the usefulness of these probes for restriction fragment length polymorphism (RFLP) linkage studies in sheep. Two-thirds of the cattle cDNA probes showed moderate to strong homology with sheep DNA samples, compared with less than half of the human cDNA probes at the final washing stringency chosen for the experiments. The set of probes tested detected a useful frequency of RFLPs. Fifty-seven per cent of probes showing moderate to strong homology identified RFLPs with one or more restriction enzymes. Restriction enzymes that detected RFLPs most frequently in sheep were Taq I and Msp I. The results show that sheep and cattle cDNA probes, including candidate genes for production traits, identified a high frequency of RFLPs suitable for genetic mapping in sheep.     

Functional characterization of a water channel of the nematode Caenorhabditis elegans

A genome project for the species Caenorhabditis elegans has demonstrated the presence of eight cDNAs belonging to the major intrinsic protein (MIP) family. We previously characterized one of these cDNAs known as C01G6.1. C01G6.1 was confirmed to be a water channel and newly designated as AQP-CE1 [Am. J. Physiol. 275 (1998) C1459-C1464]. In this paper, we examined the function of another MIP protein encoded by F40F9.9. This cDNA encodes a 274-amino acid protein showing a high sequence identity with mammalian aquaporin-8 (AQP8) water channel (35%) and d-TIP (34%), an AQP of Arabidopsis. The expression of F40F9.9 in Xenopus oocytes increased the osmotic water permeability (P(f)) 10.4-fold, and the activation energy for P(f) from Arrhenius plot was 4.7 kcal/mol, suggesting that F40F9.9 is a water channel (AQP-CE2). AQP-CE2 was not permeable to glycerol or urea. Oocyte P(f) was reversibly inhibited by 58% after an incubation with 0.3 mM HgCl(2). To identify the mercury-sensitive site, four individual cysteine residues in AQP-CE2 (at positions 47, 132, 149, 259) were altered to serine by site-directed mutagenesis. Of these mutants, only C132S had a P(f) similar to that of the wild-type together with an acquired mercury resistance, suggesting that Cys-132 is the mercury-sensitive site. Similar results were obtained by the mutation of Cys-132 to alanine (C132A). Replacement of Cys-132 with tryptophan decreased P(f) by 64%, but P(f) was still 2.5 times higher than that of the control. Cys-132 is located in the transmembrane helix 3, close to the transition to the extracellular loop C. These results suggest that the transmembrane helix 3, including Cys-132, might participate in the aqueous pore formation, or, alternatively, that Cys-132 might contribute to the construction of the AQP protein.     

Erratum: James F. O'Connell and Jim Allen (1998) when did humans first arrive in Australia and why is it important to know? Evol Anthropol 6:132–146.

James F. O'Connell and Jim Allen (1998) When Did Humans First Arrive in Australia and Why Is It Important to Know? Evol Anthropol 6:132–146. In Table 2, page 141, in column 4, the number of dates for Nunamira should have been 2.     

Assignment of linkage groups to pea chromosomes after karyotyping and gene mapping by fluorescent in situ hybridization

Chromosomes of the pea (Pisum sativum L.) were submitted to fluorescent in situ hybridization (FISH) with probes specific for the oligonucleotides (AG)₁₂, (AC)₁₂, (GAA)₁₀, and (GATA)₇ and for the genes encoding 25S rRNA, 5S rRNA and the storage proteins legumin A, K and vicilin. A fourth 5S rRNA gene locus, apparently specific for an accession of the cultivar Grüne Victoria, was newly detected. This allowed all seven chromosome pairs to be distinguished by FISH signals of rRNA genes. The same was possible using a combination of oligonucleotide probes or of oligonucleotides and rRNA gene-specific probes in multicolour FISH. Rehybridization with the 5S rRNA gene-specific probe allowed us to assign vicilin genes to the short arm of chromosome 5, the single legumin A locus to the long arm of chromosome 3 and the legumin B-type genes (exemplified by legumin K) to one locus on the short arm of chromosome 6. Correlation of these data with an updated version of the pea genetic map allowed the assignment of most linkage groups to defined chromosomes. It only remains to be established which of linkage groups IV and VII corresponds to the satellited chromosomes 4 or 7, respectively. Received: 13 February 1998; in revised form: 3 April 1998 / Accepted: 7 April 1998     

In situ localization with digoxigenin-labelled probes of tau-related mRNAs in the rat pancreas

Summary Two cDNA probes complementary to fetal rat brain tau cDNA were produced by the polymerase chain reaction (PCR) and labelled by digoxigenin-11-dUTP incorporation during the PCR elongation step. These probes were tested for thein situ localization of tau mRNAs in sections of rat cerebellum. The hybridization signal was consistent with the known localization of brain tau mRNAs, showing the validity of cDNA probes labelled by digoxigenin during the PCR. Using these probes, anin situ hybridization protocol was established and optimized for the localization of tau-related mRNAs in sections of pancreas. The aim was to determine whether these mRNAs were expressed in the exocrine or the endocrine part of the pancreas. A positive signal was found only in the exocrine part of the pancreas, and was distributed exclusively in the cytoplasm of acinar cells. The results described here are the first evidence for a specific expression of tau-related proteins in the exocrine pancreas.     

Identification of two cytosolic ascorbate peroxidase cDNAs from soybean leaves and characterization of their products by functional expression in E. coli

Screening of a cDNA library from soybean (Glycine max (L.) Merr. cv. Century) with probes based upon cytosolic ascorbate peroxidase (APx; EC 1.11.1.11) genes identified two full-length clones (SOYAPx1, SOYAPx2) apparently encoding for different soybean leaf cytosolic APxs. The deduced amino acid sequences of the two APx cDNA products differed in 13 of the 250 amino acids. The SOYAPx1 cDNA was identical to the cytosolic APx cDNA previously found in soybean root nodules. Escherichia coli expression systems were developed using both soybean APx cDNAs. Recombinant SOYAPx1 and SOYAPx2 were then utilized to characterize the enzymatic properties of the two APx cDNA products. Received: 10 May 1997 / Accepted: 19 June 1997     

- and β-tubulin mRNAs of Trypanosoma cruzi originate from a single multicistronic transcript

The cluster of alternated - and β-tubulin genes in the genome of Trypanosoma cruzi was shown to be transcribed into a single RNA molecule which upon processing gives rise to the mature - and β-tubulin mRNAs. This conclusion was based on: (i) nuclear RNA species with the same molecular mass hybridize to both - and β-tubulin cDNA probes; (ii) S₁ nuclease assay of the clustered tubulin genes has shown protected DNA fragments of the same size and of greater molecular mass than that corresponding to the mRNAs, hybridizable to both - and β-tubulin cDNA probes; (iii) β-tubulin hybrid selected RNA is still able to hybridize to -tubulin probe.