Translation and M1 double-stranded RNA propagation: MAK18 = RPL41B and cycloheximide curing.

MAK18 is one of nearly 30 chromosomal genes of Saccharomyces cerevisiae necessary for propagation of the killer toxin-encoding M1 double-stranded RNA satellite of the L-A double-stranded RNA virus. We have cloned and sequenced MAK18 and find that it is identical to RPL41B, one of the two genes encoding large ribosomal subunit protein L41. The mak18-1 mutant is deficient in 60S subunits, which we suggest results in a preferential decrease in translation of viral poly(A)-deficient mRNA. We have reexamined the curing of M1 by low concentrations of cycloheximide (G. R. Fink and C. A. Styles, Proc. Natl. Acad. Sci. USA 69:2846-2849, 1972), which is known to act on ribosomal large subunit protein L29. We find that when M1 is supported by L-A proteins made from the poly(A)+ mRNA of a cDNA clone of L-A, cycloheximide does not decrease the M1 copy number, consistent with our hypothesis.     

Yeast virus propagation depends critically on free 60S ribosomal subunit concentration.

Over 30 MAK (maintenance of killer) genes are necessary for propagation of the killer toxin-encoding M1 satellite double-stranded RNA of the L-A virus. Sequence analysis revealed that MAK7 is RPL4A, one of the two genes encoding ribosomal protein L4 of the 60S subunit. We further found that mutants with mutations in 18 MAK genes (including mak1 [top1], mak7 [rpl4A], mak8 [rpl3], mak11, and mak16) had decreased free 60S subunits. Mutants with another three mak mutations had half-mer polysomes, indicative of poor association of 60S and 40S subunits. The rest of the mak mutants, including the mak3 (N-acetyltransferase) mutant, showed a normal profile. The free 60S subunits, L-A copy number, and the amount of L-A coat protein in the mak1, mak7, mak11, and mak16 mutants were raised to the normal level by the respective normal single-copy gene. Our data suggest that most mak mutations affect M1 propagation by their effects on the supply of proteins from the L-A virus and that the translation of the non-poly(A) L-A mRNA depends critically on the amount of free 60S ribosomal subunits, probably because 60S association with the 40S subunit waiting at the initiator AUG is facilitated by the 3' poly(A).     

Dominant chromosomal mutation bypassing chromosomal genes needed for killer RNA plasmid replication in yeast

Yeast strains carrying a double-stranded RNA plasmid of 1.4–1.7 x 10⁶ daltons encapsulated in virus-like particles secrete a toxin that kills strains lacking this plasmid. The plasmid requires at least 24 chromosomal genes (pets, and mak1 through mak23) for its replication or maintenance. We have detected dominant Mendelian mutations (called KRB1 for killer replication bypass) that bypass two chromosomal genes, mak7 and pets, normally needed for plasmid replication. Strains mutant in mak7 and carrying the bypass mutation (mak7–1 KRB1) are isolated as frequent K⁺R⁺ sectors of predominantly K^-R ^- segregants from crosses of mak7–1 with a wild-type killer. All KRB1 mutations isolated in this way are inherited as single dominant centromere-linked chromosomal changes. They define a new centromere. KRB1 is not a translational suppressor. KRB1 strains contain a genetically normal killer plasmid and ds RNA species approximately the same in size and amount as do wild-type killers. Bypass of both mak7 and pets by one mutation suggests that these two genes are functionally related.

Two properties of the inheritance of KRB1 indicate an unusually high reversion frequency: (1) Heat or cycloheximide (treatments known to cure strains of the wild-type killer plasmid) readily induce conversion of mak7–1 KRB1 strains from killers to nonkillers with concomitant disappearance of KRB1 as judged by further crosses, and (2) mating two strains of the type mak7–1 KRB1 with each other yields mostly 2 K⁺R⁺: 2 K^-R^- segregation, although the same KRB1 mutation and the same killer plasmid are present in both parents.

     

Human perforin (PRF1) maps to 10q22, a region that is syntenic with mouse chromosome 10.

Perforin (PRF1) is a cytolytic, channel-forming protein of cytolytic T cells, natural killer cells, and granulated metrial gland cells and plays a crucial role in the killer cell-mediated elimination of virally infected host cells, tumor cells, and allotransplants. Two-thirds of the perforin sequence is homologous to the lytic, channel-forming complement proteins C6, C7, C8 alpha, C8 beta, and C9. Using cosmid DNA containing the PRF1 gene as a probe for fluorescence in situ hybridization, we have reevaluated its chromosomal location. Previously assigned to chromosome 17q11-q21, it has now been mapped to 10q22. The human PRF1 locus lies within a conserved synteny segment present on mouse chromosome 10, consistent with the previous chromosomal assignment of mouse perforin. The perforin locus is not linked to any of the genes of the terminal complement system.     

Physical map of a conditionally dispensable chromosome in Nectria haematococca mating population VI and location of chromosome breakpoints

Certain isolates of the plant pathogenic fungus Nectria haematococca mating population (MP) VI contain a 1.6-Mb conditionally dispensable (CD) chromosome carrying the phytoalexin detoxification genes MAK1 and PDA6-1. This chromosome is structurally unstable during sexual reproduction. As a first step in our analysis of the mechanisms underlying this chromosomal instability, hybridization between overlapping cosmid clones was used to construct a map of the MAK1 PDA6-1 chromosome. The map consists of 33 probes that are linked by 199 cosmid clones. The polymerase chain reaction and Southern analysis of N. haematococca MP VI DNA digested with infrequently cutting restriction enzymes were used to close gaps and order the hybridization-derived contigs. Hybridization to a probe extended from telomeric repeats was used to anchor the ends of the map to the actual chromosome ends. The resulting map is estimated to cover 95% of the MAK1 PDA6-1 chromosome and is composed of two ordered contigs. Thirty-eight percent of the clones in the minimal map are known to contain repeated DNA sequences. Three dispersed repeats were cloned during map construction; each is present in five to seven copies on the chromosome. The cosmid clones representing the map were probed with deleted forms of the CD chromosome and the results were integrated into the map. This allowed the identification of chromosome breakpoints and deletions.     

Genetic structure and regulation of a replicon of plasmid lambdadv.

Strains of Saccharomyces cerevisiae carrying a small double-stranded RNA species (the killer plasmid) secrete a toxin which is lethal only to strains not carrying this plasmid.We have isolated mutants in eight chromosomal genes essential for replication or maintenance of the killer plasmid, called mak1 through mak8. Seven of these genes have been mapped. mak4 and mak5 are on chromosome II; mak1 and mak8 are on chromosome XV; mak3 and mak6 are on chromosome XVI; and mak7 is on chromosome VIII. We have not yet located mak2. Two other chromosomal genes, m and pets, have been shown to be required for replication or maintenance of the killer plasmid.One allele of mak1 results in temperature sensitivity for host growth. Two independent pets isolates also result in the petite phenotype, as well as temperature sensitivity for growth.Wild-type killer strains have been reported to carry two species of doublestranded RNA of 2.5 × 10⁶ and 1.4 × 10⁶ molecular weight (designated L and M, respectively); wild-type non-killers carried only L. We estimate the size of the L and M species at 3.0 × 10⁶ and 1.7 × 10⁶ daltons, respectively. We have also detected a third species of double-stranded RNA of molecular weight 3.8 × 10⁶ (XL) present in all killer and non-killer strains examined.Mutation of any of mak1 through mak8 results in loss of the killer-associated species of double-stranded RNA (M; 1.7 × 10⁶). These mutants retain both the L species (3.0 × 10⁶) and the XL species (3.8 × 10⁶) of double-stranded RNA, and have acquired two new minor RNA species.     

The MAK11 protein is essential for cell growth and replication of M double-stranded RNA and is apparently a membrane-associated protein

MAK11 is a gene necessary for the maintenance of killer M1 double-stranded RNA, but not for other cellular double-stranded RNAs (L-A, L-BC, T, W). The DNA sequence of this gene revealed a 1407-base pair open reading frame, which corresponds to a 54-kDa protein. The C-terminal region is lysine-rich and is necessary for mak11-complementing activity. The N-terminal 24 amino acids of the open reading frame include 16 hydrophobic amino acids, 4 basic residues, and 4 neutral amino acids; this sequence could span a membrane. We constructed a MAK11-lacZ fusion that includes the entire MAK11 protein and complements the mak11-1 mutation. The fusion protein was localized in a membrane fraction as shown by centrifugation in Percoll gradients. The fusion protein could be released from the membrane fraction by salt washing. Western blotting of protein, isolated from the membrane fraction and purified by p-aminophenyl-beta-D-thiogalactoside-agarose column chromatography, revealed a fusion protein monomer of 170 kDa which agrees with the predicted molecular weight. While the mak11-1 mutation results in specific loss of M1 double-stranded RNA without any apparent growth defect, replacing a 792-base pair internal EcoRV fragment of MAK11 with the URA3 gene (gene disruption) resulted in a lethal mutation.     

Deletion of mitochondrial DNA bypassing a chromosomal gene needed for maintenance of the killer plasmid of yeast

Strains of Saccharomyces cerevisiae carrying a 1.4 x 10⁶ dalton double-stranded (ds) RNA in virus-like particles (the killer plasmid or virus) secrete a toxin that is lethal to strains not carrying this plasmid (virus). The mak10 gene is one of 24 chromosomal genes (called pets, mak1, mak2,...) that are needed to maintain and replicate the killer plasmid. We report here isolation of spontaneous and induced mutants in which the killer plasmid is maintained and replicated in spite of a defect in the mak10 gene. The bypass (or suppressor) mutations in these strains are in the mitochondrial genome. Respiratory deficiency produced by various chromosomal pet mutations, by chloramphenicol, or by antimycin A, does not bypass the mak10–1 mutation. Several spontaneous mak10–1 killer strains have about 12-fold more of the killer plasmid ds RNA than do wild-type killers. Although the absence of mitochondrial DNA bypasses mak10–1, it does not bypass pets–1, mak1–1, mak3–1, mak4–1, mak5–1, mak6–1, mak7–1, or mak8–1.     

Isolation of a cDNA clone for the human laminin-B1 chain and its gene localization.

A cDNA clone encoding the B1 chain of human laminin has been isolated from a human endothelial cell cDNA library. With use of this probe and a panel of rodent/human somatic-cell hybrids and in situ hybridization, the gene for the human laminin-B1 chain has been localized to chromosome 7, band q31.     

Assignment by in situ hybridization of the angiotensinogen gene to chromosome band 1q4, the same region as the human renin gene

Summary A 1.8kb human cDNA probe for angiotensinogen (renin substrate) was used to determine the chromosomal location of the angiotensinogen gene by in situ hybridization. The results show that human chromosome region 1q4 contains the angiotensinogen gene. The human renin gene has also recently been assigned to the same band of chromosome 1. Thus, the angiotensinogen and renin genes are located in the same region of chromosome 1.     

Human Fas Associated Factor 1, hFAF1, Gene Maps to Chromosome Band 1p32

Human Fas associated factor 1 protein (hFAF1) is involved in the positive regulation of Fas signaling even though it can not initiate the signal for itself. By chromosomal assignment using somatic cell hybrids (CASH), the hFAF1 gene was located on human chromosome 1 between markers D1S443 and D1S197. The hFAF1 gene was mapped to human chromosome band 1p32 by FISH utilizing a genomic PAC clone containing the gene. In genomic Southern analysis using hFAF1 cDNA as a probe, several bands appeared in three different restriction enzyme digestions. The single band appearance in FISH analysis compared to several bands in Southern blots implies that the hFAF1 gene would be rather big or that an additional hFAF1 gene isotype(s) might be present in close vicinity.     

Suppression of chromosomal mutations affecting M1 virus replication in Saccharomyces cerevisiae by a variant of a viral RNA segment (L-A) that encodes coat protein.

For the maintenance of "killer" M1 double-stranded RNA in Saccharomyces cerevisiae, more than 30 chromosomal genes are required. The requirement for some of these genes can be completely suppressed by a cytoplasmic element, [B] (for bypass). We have isolated a mutant unable to maintain [B] (mab) and found that it is allelic to MAK10, one of the three chromosomal MAK genes required for the maintenance of L-A. The heat curing of [B] always coincided with the loss of L-A. To confirm that [B] is located on L-A, we purified viral particles containing either L-A or M1 from strains with or without [B] activity and transfected these purified particles into a strain which did not have either L-A or M1. The transfectants harboring L-A and M1 from a [B] strain showed the [B] phenotype, but the transfectants with L-A and M1 from a [B-o] strain did not show the [B] phenotype. Furthermore, the transfectants having L-A from a [B] strain and M1 from a [B-o] strain also showed the [B] phenotype. Therefore, we concluded that [B] is a property of a variant of L-A. In the transfection experiment, we also proved that the superkiller phenotype of the [B] strain is a property of L-A and that L-A with [B] activity can maintain a higher copy number of M1 regardless of the source of M1 viruslike particles. These data suggest that MAK genes whose mutations are suppressed by [B] are concerned with the protection of M1 (+) single-stranded RNA or the formation of M1 viruslike particles and that an L-A with more efficient production of M1 viruslike particles can completely dispense with the requirement for those MAK genes.     

Localization of the human salivary protein complex (SPC) to chromosome band 12p13.2

In situ hybridization of a 3H-labeled probe containing a fragment from PRP-1, a genomic clone with human salivary proline-rich protein gene sequences, revealed significant labeling on the short arm of human chromosome 12 in metaphase preparations from two individuals. Fifty-three percent of metaphases exhibited labeling on one or both chromosomes 12. Additional cells scored at the 850-1,000 band level revealed a significant proportion (52% [32/61] grains, p less than 0.005) of the labeled sites on chromosome 12 to be on band 12p13.2. This probe for a human salivary proline-rich protein gene fragment, probably PMS, is from a cluster of 13 linked genes designated as the human salivary protein complex (SPC). Studies of the DNA of human-mouse somatic-cell hybrids have assigned the SPC to chromosome 12, but have not provided a regional localization (Azen et al, 1985). This paper reports the localization of the SPC to a specific chromosomal band, 12p13.2.     

Acute megakaryocytic leukaemia with acquired polysomy 21 and translocation t(1;21)

Cytogenetic study of a young child with acute megakaryocytic leukaemia (AML-M7) has shown a karyotype with 49-50 chromosomes with one and two acquired extra chromosomes 21. Fluorescence in situ hybridization detected a minor clone with translocation t(1;21) and loss of a part of chromosome band 1p36. Trisomy and polysomy 21 are not uncommon in AML-M7. A more systematic search for chromosome 21 rearrangements in AML-M7 using FISH techniques is proposed.     

The molecular organisation of a B chromosome tandem repeat sequence fromBrachycome dichromosomatica

A high copy, tandemly repeated, sequence (Bd49) specific to the B chromosome and located near the centromere in Brachycome dichromosomatica was used to identify lambda genomic clones from DNA of a 3B plant. Only one clone of those analysed was composed entirely of a tandem array of the B-specific repeat unit. In other clones, the Bd49 repeats were linked to, or interspersed with, sequences that are repetitious and distributed elsewhere on the A and B chromosomes. One such repetitious flanking sequence has similarity to retrotransposon sequences and a second is similar to chloroplast DNA sequences. Of the four separate junctions analysed of Bd49-like sequence with flanking sequence, three were associated with the same A/T-rich region in Bd49 and the fourth was close to a 25 bp imperfect dyadic sequence. No novel B-specific sequences were detected within the genomic clones. Received: 31 December 1995; in revised form: 1 May 1996 / Accepted: 10 May 1996     

Comparison of two Xanthomonas campestris pathovar campestris genomes revealed differences in their gene composition

For the Xanthomonas campestris pathovar campestris wild-type strain B100 a plasmid-based clone library was constructed. The plasmids carried chromosomal fragments of 3-4 kb in size that were tagged in vitro with the artificial transposon KAN-2. More than 3000 of the transposon target sites were characterized by DNA sequencing. The sequences obtained were compared to the recently published genome of Xanthomonas campestris pathovar campestris strain ATCC 33913. Most of the sequenced clones derived from strain B100 matched the chromosomal sequence of strain ATCC 33913. An alignment to the circular map of this chromosome revealed that the similarities were statistically distributed over the entire genome of strain ATCC 33913. The similarity was obvious for protein coding sequences, as well as for mobile genetic elements. However, four regions in the genome of Xanthomonas campestris pathovar campestris strain ATCC 33913, ranging in size from 11 to 37 kb, were not represented in the sequenced clone library of Xanthomonas campestris pathovar campestris strain B100. On the other hand, 1.2% of the sequenced clones originating from Xanthomonas campestris pathovar campestris strain B100 showed no or insignificant similarities to the genome of strain ATCC 33913.     

Localization of the beta A4-crystallin gene (CRYBA4) on human chromosome 22 in the region q11.2-->q13.1.

The chromosomal localization of the human gene (CRYBA4) coding for the eye lens protein beta A4-crystallin has been carried out using a nearly full-length cDNA clone encoding bovine beta A4-crystallin. A panel of 21 human-mouse or human-hamster hybrid cell lines derived from different parental combinations was characterized with respect to the human chromosomal content and the presence of well established human chromosome-specific markers. These panels were screened for the presence of CRYBA4 using the bovine cDNA clone as a probe. A 100 percent concordance was observed between the presence or absence of the CRYBA4 and human chromosome 22 indicating that the gene resides on this chromosome. By using cell hybrids containing translocated chromosome 22 segments, the localization could be refined to the region 22q11.2-->q13.1.     

Evidence for a new chromosome in Saccharomyces cerevisiae.

The current yeast map has 16 chromosomes, each originally defined by a centromere-linked gene unlinked to previously defined centromere markers. We examined four genes, cly2, KRB1, AMY2, and tsm0115, each centromere linked, but previously thought to be not on chromosomes I to XVI. We found that AMY2 is linked to cly2, and both are on chromosome II. tsm0115 is on the left arm of chromosome XVI. We confirm the earlier evidence that KRB1 is not on chromosomes I through XVI. This gene thus defines a new chromosome XVII. We also report meiotic linkage of met4 and pet8 (on chromosome XIV), confirming the connection between the petx-kex2 fragment of XIV and the centromere of XIV.     

The mouse alpha 1(XII) and human alpha 1(XII)-like collagen genes are localized on mouse chromosome 9 and human chromosome 6.

Type XII collagen is a member of the FACIT (fibril-associated collagens with interrupted triple helices) group of extracellular matrix proteins. Like the other members of this group, collagen types IX and XIV, type XII has alternating triple-helical and non-triple-helical domains. Because of its structure, its association with collagen fibrils, and its distribution in dense connective tissues, type XII is thought possibly to act as a cross-bridge between fibrils and resist shear forces caused by tension. A portion of the ffuse gene was isolated by screening a genomic library with a chicken alpha 1 (XII) cDNA probe, followed by subcloning and sequence analysis. Comparison of exon sequences with the sequence of a mouse cDNA clone allowed the mouse gene to be identified as the alpha 1 (XII) collagen gene. In the mouse, Col12a1 is located on chromosome 9, as determined by linkage analysis using DNA from interspecific backcrosses with Mus spretus. Screening of a human genomic library also allowed the isolation of a human alpha 1(XII)-like gene (CoL12A1). This gene was mapped to chromosome 6 by blot hybridization to DNA from human/hamster hybrid cell lines. This information should prove useful in determining the role of type XII collagen genes as candidate genes in inheritable connective tissue diseases.