Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti)

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human Mhc DRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB088371–AB088374, AB089199, AB154393–AB154399, and AB162144.     

Analysis of zebrafish Mhc using BAC clones

 Although major histocompatibility complex (Mhc) genes have been identified in a number of species, little is yet known about their organization in species other than human and mouse. The zebrafish, Danio rerio, is a good candidate for full elucidation of the organization of its Mhc. As a step toward achieving this goal, a commercially available zebrafish BAC library was screened with probes specific for previously identified zebrafish class I and class II genes, as well as for genes controlling the proteasome subunits LMP7 and LMP2. Restriction maps of the individual positive clones were prepared and the Mhc (LMP7) genes localized to specific fragments. The total length of genomic DNA fragments with Mhc genes was approximately 1700 kilobases (kb) (200 kb of fragments bearing class I loci and 1500 kb of fragments bearing class II loci). One of the two class I loci (Dare-UCA) is closely associated with the LMP7 locus; the second class I locus (Dare-UAA) is more than 50 kb distant from the UCA locus and has no LMP genes associated with it. None of the class II genes are linked to the class I or the LMP genes. All six of the previously identified class II B genes and one of the three class II A genes were found to be present in the BAC clones; no new Mhc loci could be identified in the library. Each of the six previously identified class II B loci was found to be borne by a separate group of BAC clones. The Dare-DAB and -DAA loci were found on the same clone, approximately 15 kb apart from each other. An expansion of DCB and DDB loci was detected: the zebrafish genome may contain at least five closely related DCB and two closely related DDB loci which are presumably the products of relatively recent tandem duplication. These results are consistent with linkage studies and indicate that in the zebrafish, the class I and class II loci are on different chromosomes, and the class II loci are in three different regions, at least two of which are on different chromosomes. Received: 14 August 1997 / Revised: 16 September 1997     

Analysis of the occurrence and nature of repeated DNA in an 850 kb region of Arabidopsis thaliana chromosome 4

The occurrence and nature of repeated DNA sequences has been analysed within an 850 kb YAC contig on Arabidopsis thaliana chromosome 4. Hybridization analysis with seven RFLP markers, six cosmid contigs, 29 YAC end probes and eight YAC clones showed that a least 585 kb of the 850 kb contained only low-copy sequences. One YAC end probe, EG15C8LE, hybridized to multiple genomic fragments and contained a sequence with predicted protein homology to cytochrome P450 monooxygenases. Another one, EG11B7RE, was found to be non-contiguous with the other YAC clones and contained a dispersed repetitive sequence associated with centromeric regions     

Chromosome Walking in the Petunia Inflata Self-Incompatibility (S-) Locus and Gene Identification in an 881-kb Contig Containing S 2 -RNase

Self-incompatibility (SI) in the Solanaceae, Rosaceae and Scrophulariaceae is controlled by the polymorphic S locus, which contains two separate genes encoding pollen and pistil determinants in SI interactions. The S-RNase gene encodes the pistil determinant, whereas the pollen determinant gene, named the pollen S gene, has not yet been identified. Here, we set out to construct an integrated genetic and physical map of the S locus of Petunia inflata and identify any additional genes located at this locus. We first conducted chromosome walking at the S2 locus using BAC clones that contained either S2-RNase or one of the nine markers tightly linked to the S locus. Ten separate contigs were constructed, which collectively spanned 4.4 Mb. To identify additional genes located at the S2 locus, a 328-kb region (part of an 881-kb BAC contig) containing S2-RNase was completely sequenced. Approximately 76% of the region contained repetitive sequences, including transposon-like sequences. Other than S2-RNase, an F-box gene, named PiSLF2 (S2-allele of P. inflata S-locus F-box gene), was the only predicted gene whose deduced amino acid sequence was similar to the sequences of known proteins in the database. Two different cDNA selection methods were used to identify additional genes in the 881-kb contig; 11 groups of cDNA clones were identified in addition to those for S2-RNase and PiSLF2. RT-PCR analysis of expression profiles and PCR analysis of BAC clones and genomic DNA confirmed that seven of these 11 newly identified genes were located in the 881-kb contig.     

Gene organization of the quail major histocompatibility complex (MhcCoja) class I gene region

 Class I genomic clones of the quail (Coturnix japonica) major histocompatibility complex (MhcCoja) were isolated and characterized. Two clusters spanning the 90.8 kilobase (kb) and 78.2 kb class I gene regions were defined by overlapping cosmid clones and found to contain at least twelve class I loci. However, unlike in the chicken Mhc, no evidence for the existence of any Coja class II gene was obtained in these two clusters. Based on comparative analysis of the genomic sequences with those of the cDNA clones, Coja-A, Coja-B, Coja-C, and Coja-D (Shiina et al. 1999), these twelve loci were assigned to represent one Coja-A gene, two Coja-B genes (Coja-B1 and -B2), four Coja-C genes (Coja-C1-C4), four Coja-D genes (Coja-D1-D4), and one new Coja-E gene. A class I gene-rich segment of 24.6 kb in which five of these genes (Coja-B1, -B2, -D1, -D2 and -E) are densely packed were sequenced by the shotgun strategy. All of these five class I genes are very compact in size [2089 base pairs (bp)–2732 bp] and contain no apparent genetic defect for functional expression. A transporter associated with the antigen processing (TAP) gene was identified in this class I gene-rich segment. These results suggest that the quail class I region is physically separated from the class II region and characterized by a large number of the expressible class I loci (at least seven) in contrast to the chicken Mhc, where the class I and class II regions are not clearly differentiated and only at most three expressed class I loci so far have been recognized. Received: 9 March 1998 / Revised: 12 October 1998     

An approach to cloning of Pi-b rice blast resistance gene

A contig of clones from BAC rice genomic library encompassing blast resistance gene Pi-b was constructed. On an average eight clones (8 ± 2.6) were picked up by each marker, which was expected basing on the BAC library size (Nakamura et al. 1997). The 2.4 cM distance between flanking RFLP markers G 1234 and RZ 213 (Miyamoto et al. 1996) was spanned with 4 steps of contig including 25 clones. The physical distance of 370 kb between flanking markers corresponds to a small ratio of physical and genetical distances (155 kb/cM) due to a probable structure of the gene locus near the telomeric end of the chromosome. Markers cosegregating with blast resistance against Magnoporthe grisea were localized in a 2 kb restriction fragment. A new border marker was found on the telomeric side of the Pi-b gene, less than 10 kb from cosegregating markers. No clear marker for the centromeric side of the gene was found but the position of Pi-b rice blast resistant gene was narrowed to within at least 50 kb, which is to our knowledge the most precised estimation of the position of this gene.     

Cloning and tissue expression of cDNAs from chromosome 5q21–22 which is frequently deleted in advanced lung cancer

Previously, we have reported that the inactivation of putative tumor-suppressor gene(s) on chromosome 5q21–22 may play an important role in the progression of lung cancer. Here, we describe the establishment of a yeast artificial chromosome (YAC) contig that spans 8–10 Mb at the 5q21–22 region. Six cosmid contigs have also been established in this YAC contig. About 35 exon-like fragments have been detected by exon-amplification, direct screening, cross-species hybridization, and searches of a database. Thus far, 14 cDNAs have been isolated, and two of them coincide with known genes, viz., cysteine dioxygenase I and geranylgeranyltransferase I. The other 12 cDNAs are considered to be novel genes. Two of these novel cDNA show partial homology to known genes, viz., semaphorin CD100 and the 28S rRNA gene. In addition, four known genes, including APC (adenomatous polyposis coli), MCC (mutated in colorectal cancer), proto-oncogene tyrosine kinase FER, and genomic imprinted gene U2AF1-RS1, have also been mapped in this contig. This large contig and expression map should prove crucial in the identification of susceptibility gene(s) related to the progression of lung cancer. Received: 27 May 1997 / Accepted: 3 September 1997     

Comparison between a coffee single copy chromosomal region and Arabidopsis duplicated counterparts evidenced high level synteny between the coffee genome and the ancestral Arabidopsis genome

The Arabidopsis thaliana genome sequence provides a catalogue of reference genes that can be used for comparative analysis of other species thereby facilitating map-based cloning in economically important crops. We made use of a coffee bacterial artificial chromosome (BAC) contig linked to the S_H3 leaf rust resistance gene to assess microsynteny between coffee (Coffea arabica L.) and Arabidopsis. Microsynteny was revealed and the matching counterparts to C. arabica contigs were seen to be scattered throughout four different syntenic segments of Arabidopsis on chromosomes (Ath) I, III, IV and V. Coffee BAC filter hybridizations were performed using coffee putative conserved orthologous sequences to Arabidopsis predicted genes located on the different Arabidopsis syntenic regions. The coffee BAC contig related to the S_H3 region was successfully consolidated and later on validated by fingerprinting. Furthermore, the anchoring markers appeared in same order on the coffee BAC contigs and in all Arabidopsis segments with the exception of a single inversion on AtIII and AtIV Arabidopsis segments. However, the S_H3 coffee region appears to be closer to the ancestral genome segment (before the divergence of Arabidopsis and coffee) than any of the duplicated counterparts in the present-day Arabidopsis genome. The genome duplication events at the origin of its Arabidopsis counterparts occurred most probably after the separation (i.e. 94 million years ago) of Euasterid (Coffee) and Eurosid (Arabidopsis).     

Construction and application of a bacterial artificial chromosome (BAC) library of<Emphasis Type="Italic"> Prunus armeniaca</Emphasis> L. for the identification of clones linked to the self-incompatibility locus

To facilitate gene discovery in the Rosaceae, a bacterial artificial chromosome (BAC) library was constructed using high-molecular-weight (HMW) DNA from apricot leaves ( Prunus armeniaca L.). The library contains 101,376 clones (264, 384-well plates) with an average insert size of 64 kb, equivalent to 22-fold genome coverage. In the first application of this library, high-density filters were screened for self-incompatibility genes using apricot DNA probes. Eight positive BAC clones were detected and fingerprinted to determine clone relationships and assemble contigs. These results demonstrate the suitability of this library for gene identification and physical mapping of the apricot genome.Communicated by R. Hagemann     

Construction and Characterization of aMagnaporthe griseaBacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of aMagnaporthe griseabacterial artificial chromosome library.Fungal Genet. Biol.20,280–288. A bacterial artificial chromosome (BAC) library ofMagnaporthe griseacontaining 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents ofM. griseaand has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using alys1-3auxotroph, we have shown that BAC clones at least 113 kb can be transformed intoM. griseato screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

Genetic and physical mapping of xa13, a recessive bacterial blight resistance gene in rice

 The recessive gene, xa13, confers resistance to Philippine race 6 (PXO99) of the bacterial blight pathogen Xanthomonas oryzae pv oryzae. Fine genetic mapping and physical mapping were conducted as initial steps in an effort to isolate the gene. Using nine selected DNA markers and two F₂ populations of 132 and 230 plants, xa13 was fine-mapped to a genomic region <4 cM on the long arm of rice chromosome 8, flanked by two RFLP markers, RG136 and R2027. Four DNA markers, RG136, R2027, S14003, and G1149, in the target region were used to identify bacterial artificial chromosome (BAC) clones potentially harboring the xa13 locus from a rice BAC library. A total of 11 BACs were identified, forming four separate contigs including a single-clone contig, 29I3, associated with the RG136 STS marker, the S14003 contig consisting of four clones (44F8, 41O2, 12A16, and 12F20), the G1149 contig with two clones, 23D11 and 21H18, and the R2027 contig consisting of four overlapping clones, 42C23, 30B5, 6B7 and 21H14. Genetic mapping indicated that the xa13 locus was contained in the R2027 contig. Chromosomal walking on the R2027 contig resulted in two more clones, 33C7 and 14L3. DNA fingerprinting showed that the six clones of the R2027 contig were overlapping. Clone 44F8 hybridized with a single fragment from the clone 14L3, integrating the R2027 and S14003 contigs into a single contig consisting of ten BAC clones with a total size of approximately 330 kb. The physical presence of the xa13 locus in the contig was determined by mapping the ends of the BAC inserts generated by TAIL-PCR. In an F₂ population of 230 plants, the BAC-end markers 42C23R and 6B7F flanked the xa13 locus. The probes 21H14F and 21H14R derived from BAC clone 21H14 were found to flank xa13 at a distance of 0.5 cM on either side, using a second F₂ population of 132 plants. Thus, genetic mapping indicated that the contig and the 96-kb clone, 21H14, contained the xa13 locus. Received: 15 August 1998 / Accepted: 29 September 1998     

A physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome

A combined physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome was constructed using pulsed-field gel electrophoresis (PFGE) and hybridizations with cloned gene probes. Total genomic DNA was digested with the meganucleasesSwaI (5-ATTTAAAT-3),PacI (5-TTAATTAA-3), andPmeI (5-GTTTAAAC-3) yielding 26, 27, and 23 fragments, respectively. The chromosomal restriction fragments were then separated by PFGE. By summing up the lengths of the fragments generated with each of the three enzymes, a genome size of 3082 +/- 20 kb was determined. To identify adjacentSwaI fragments, a genomic cosmid library ofC. glutamicum was screened for chromosomal inserts containingSwaI sites. Southern blots of the PFGE gels were hybridized with these linking clones to connect theSwaI fragments in their natural order. By this method, about 90% of the genome could be ordered into three contigs. Two of the remaining gaps were closed by cross-hybridization of blottedSwaI digests using as probesPacI andPmeI fragments isolated from PFGE gels. The last gap in the chromosomal map was closed by hybridization experiments using partialSwaI digestions, thereby proving the circularity of the chromosome. By hybridization of gene probes toSwaI fragments separated by PFGE about 30 genes, including rRNA operons, IS element and transposon insertions were localized on the physical map.     

BAC/YAC contigs from the H2-M region of mouse Chr 17 define gene order as Znf173-Tctex5-Mog-D17Tu42-M3-M2

 A yeast artificial chromosome (YAC) contig from the C57BL/6 (H2 ^b ) mouse was created from the major histocompatibility complex (Mhc, H2 in mouse) class Ib subregion, H2-M. It spans approximately 1.2 megabase (Mb) pairs and unites the previous >1.5-Mb YAC contigs (Jones et al. 1995) into a single contig, which includes 21 Mhc class I genes distal to H2-T1. A bacterial artificial chromosome (BAC) contig from the 129 (H2 ^bc ) mouse, spanning approximately 600 kilobases, was also built from Znf173 (Afp, a gene for acid finger protein), through Tctex5 (t-complex testis expressed-5) and Mog (myelin oligodendrocyte glycoprotein), to H2-M2. Twenty-four sequence-tagged site (STS) markers were newly developed, and 35 markers were mapped in the YAC/BAC contigs, which define the marker order as Cen –Znf173–Tctex5 – Mog–D17Tu42–D17Mit232–H2-M3–D17Leh525–H2-M2– Tel. The gene order of Znf173 – Tctex5 – Mog – D17Tu42 is conserved between mouse and human, showing that the middle H2-M region corresponds to the subregion of the human Mhc surrounding HLA-A. Received: 25 July 1997 / Revised: 10 September 1997     

Mhc class I and non-class I gene organization in the proximal H2-M region of the mouse

 A bacterial artificial chromosome (BAC) contig was constructed across the proximal part of the H2-M region from the major histocompatibility complex (Mhc) of mouse strain 129 (H2 ^bc ). The contig is composed of 28 clones that span approximately 1 megabasepair (Mb), from H2-T1 to Mog, and contains three H2-T genes and 18 H2-M genes. We report the fine mapping of the H2-M class I gene cluster, which includes the previously reported M4-M6, the M1 family, the M10 family, and four additional class I genes. All but two of the H2-M class I genes are conserved among haplotypes H2 ^k , H2 ^b , and H2 ^bc , and only two genes are found in polymorphic HindIII fragments. Six evolutionarily conserved non-class I genes were mapped to a 180 kilobase interval in the distal part of the class I region in mouse, and their order Znf173-Rfb30-Tctex5-Tctex6-Tctex4-Mog was found conserved between human and mouse. In this Znf173-Mog interval, three mouse class I genes, M6, M4, and M5, which are conserved among haplotypes, occupy the same map position as the human HLA-A class I cluster, which varies among haplotypes and is diverged in sequence from the mouse genes. These results further support the view that class I gene diverge and evolve independently between species. Received: 27 April 1998 / Revised: 4 June 1998     

Cloning and expression of the SalI restriction-modification genes of Streptomyces albus G

Summary The Streptomyces albus G genes (salR and salM) for the class II restriction enzyme SalI (SalGI) and its cognate modification enzyme were cloned in Streptomyces lividans 66. Selection was initially for the salR gene. From a library of S. albus G DNA in the high copy number plasmid pIJ486 several clones of S. lividans were obtained that were resistant to phage C31 unmodified at the many SalI sites in its DNA, but were sensitive to modified phages last propagated on a restriction-deficient, modification-proficient mutant of S. albus G. SalI activity was detected in cell-free extracts of the clones, though only at levels comparable with that in S. albus G. Five different recombinant plasmids were isolated, with inserts of 5.6, 5.7, 8.9, 10 and 18.9 kb that contained a common region of 4.5 kb. These plasmids could not be digested by SalI, although the vector has four recognition sites for this enzyme, indicating that the salM gene was also cloned and expressed. Subcloning experiments in S. lividans indicated the approximate location of salR and salM, and in Escherichia coli led to detectable expression of salM but not of salR. A variety of previously isolated S. albus G mutants affected in aspects of SalI-specific restriction and modification were complemented by the cloned DNA; they included a mutant temperature-sensitive for growth apparently because of a mutation in salM. Southern blotting showed that DNA homologous to the cloned sal genes was present in Xanthomonas and Rhodococcus strains, but not detectably in Herpetosiphon strains, all of which produce SalI isoschizomers.     

Construction of a bacterial artificial chromosome (BAC) library for citrus and identification of BAC contigs containing resistance gene candidates

A BAC library was constructed from the genomic DNA of an intergeneric Citrus and Poncirus hybrid. The library consists of 24,576 clones with an average insert size of 115 kb, representing approximately seven haploid genome equivalents and is able to give a greater than 99% probability of isolating single-copy citrus DNA sequences from this library. High-density colony hybridization-based library screening was performed using DNA markers linked to the citrus tristeza virus (CTV) resistance gene and citrus disease resistance gene candidate (RGC) sequences. Between four and eight clones were isolated with each of the CTV resistance gene-linked markers, which agrees with the library’s predicted genome coverage. Three hundred and twenty-two clones were identified using 13 previously cloned citrus RGC sequences as probes in library screening. One to four fragments in each BAC were shown to hybridize with RGC sequences. One hundred and nine of the RGC BAC clones were fingerprinted using a sequencing gel-based procedure. From the fingerprints, 25 contigs were assembled, each having a size of 120–250 kb and consisting of 2–11 clones. These results indicate that the library is a useful resource for BAC contig construction and molecular isolation of disease resistance genes. Received: 22 May 2000 / Accepted: 25 September 2000     

Organization of the ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA gene is separated from the 16S and 23S rRNA genes

Summary In order to study the organization of the ribosomal RNA genes of Mycoplasma hyopneumoniae the rRNA genes were cloned in phage vectors EMBL3 and EMBL4. By subcloning the restriction fragments into various plasmids and analysing the resulting clones by Southern and Northern blot hybridization, a restriction map of the rRNA genes was generated and the organization of the rRNA genes was determined. The results show that the genes for the 16S and 23S rRNAs are closely spaced and occur only once in the genome, whereas the 5S rRNA gene is separated from the other two genes by more than 4 kb.