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Complementary DNA encoding three catalytic subunits of protein phosphatase 1 (PP1 alpha, PP1 beta, and PP1 gamma) and the insulin-stimulated protein kinase 1 (ISPK-1) was analyzed for variations in the coding regions related to insulin-resistant glycogen synthesis in skeletal muscle of 30 patients with non-insulin-dependent diabetes mellitus (NIDDM). The human ISPK-1 cDNA was cloned from T-cell leukemia and placental cDNA libraries and mapped to the short arm of the human X chromosome. Single-strand conformation polymorphism (SSCP) analysis identified a total of six variations in the coding regions of the PP1 genes: two in PP1 alpha at codons 90 and 255; one in PP1 beta at codon 67; and three in PP1 gamma at codons 11,269, and 273, respectively. All were, however, silent single nucleotide substitutions. SSCP analysis of the ISPK-1 gene identified one silent polymorphism at codon 266 and one amino acid variant at codon 38 (Ile-->Ser). This variant was primarily found in one male NIDDM patient. This subject, however, did not exhibit an impairment of muscle insulin-stimulated glycogen synthase activation. No significant differences were found in mRNA levels in muscle of the four genes between 15 NIDDM patients and 14 healthy subjects. Our findings suggest that 1) genetic abnormalities in the coding regions of PP1 alpha, PP1 beta, PP1 gamma, and ISPK-1 are unlikely to be frequently occurring causes of the reduced insulin-stimulated activation of the glycogen synthesis in muscle from the analyzed group of NIDDM patients; 2) the mRNA levels of PP1 alpha, PP1 beta, PP1 gamma, and ISPK-1 are normal in muscle from the NIDDM patients; and 3) putative inherited defects in insulin-stimulated activation of muscle glycogen synthesis in patients with insulin-resistant NIDDM may be located further upstream of ISPK-1 in the insulin action cascade.

Investigations of myelin disorders, in particular multiple sclerosis (MS), have concentrated on immunemediated damage to formed myelin, while there has been less emphasis on the molecular genetics of myelin formation. We have generated a transgenic mouse mutant (designated 2-50) which carries an insertional mutation in a locus regulating myelination. These mice carry a transgene comprising 1.3 Kb of the mouse myelin basic protein (MBP) promoter conjugated to a fragment containing exons 2 and 3 of the human c-myc gene. Positive mice show a significant reduction in myelin compared to controls and a shivering phenotype. Unlike other myelin mutants, all 2-50 mice lose the shivering phenotype and breed normally. Expression of c-myc is detectable in only 65% of transgene-carrying mice, and when present occurs at extremely low levels. This shows that the phenotype is caused by insertional inactivation of a gene necessary for myelination rather than ectopic expression of the transgene. The transgene was found by in situ hybridization to be inserted into a single site which is very distally located on chromosome 9. The 2-50 mice represent a unique model which will be ideal for investigating the molecular basis of myelin assembly and for developing gene therapy to promote remyelination in conditions such as MS.

Two semen samples from a 47,XXY male were examined using chromosome-specific DNA probes and fluorescent in situ hybridization (FISH) to determine the distribution of sex chromosomes and an autosome (chromosome 17) in the sperm. A motile population of sperm was also prepared from one sample using the swim-up technique to compare the motile and total sperm populations. Chromosomes were localized using single FISH and a biotinylated chromosome 17 probe (TR17), or double FISH using a biotinylated X chromosome probe (TRX) and a digoxigenin-labelled Y chromosome probe (HRY). Labelling efficiencies were 95-98%. Ploidy levels were estimated by measurement against a microscope eye-piece graticule. The overall ratio of X- to Y-bearing sperm was 47% to 48.4% in the neat samples, and 48.4% to 45.3% in the swim-up fraction. Neither of the ratios was significantly different from 1:1. The frequencies of monosomic and disomic (but otherwise haploid sperm) were not different from the frequencies we observed in normal donors. In contrast, the frequencies of both diploid and tetraploid cells were increased in the neat samples of the XYY male. In the swim-up fractions, however, none of these parameters differed from those of ten normal semen donors. These results support the hypothesis that the extra Y chromosome in XYY men is eliminated during spermatogenesis.

Because of the conservation of the ubiquitin coding sequence and the number of transcriptionally active genes and reverse-transcribed pseudogenes, it has not been possible to use ubiquitin cDNA clones to map the functional ubiquitin genes. The UBB and UBC polyubiquitin genes have previously been mapped by the use of specific intron or 5' flanking sequence probes. In this study, we have used an intron sequence from the UBA52 gene for chromosome mapping studies. Analysis of somatic cell hybrids containing individual human chromosomes indicated that the UBA52 gene is located on chromosome 19. In situ hybridization studies confirmed the chromosomal localization but showed two peaks of hybridization: a major one over 19p13.1-p12 and a secondary one over 19q12-q13.11. Because the peak of hybridization over 19p13.1-p12 was consistently the strongest in five individuals, it is likely that this is the location of the UBA52 gene. Thus far, three of the four transcriptionally active ubiquitin genes have been assigned to separate chromosomes.

The chromosomal localization of the human Mu class glutathione S-transferase (GST) genes has been complicated by two factors; the total number of genes is unknown and there is a polymorphism that results from the presence or absence of the GSTM1 gene. Three human Mu class glutathione S-transferase isoenzymes, GSTM1, GSTM2, and GSTM3, have been characterized previously, and we have recently cloned and characterized GSTM4, another member of this class. Here we report that a probe derived from GSTM4 cross-hybridizes with the other three known human Mu class GST genes. In situ hybridization with the GSTM4 probe localized a major region of hybridization on chromosome band 1p13. Although there is a region of very weak hybridization on chromosome 6, these data indicate that the human Mu class gene family is largely clustered and not dispersed on different chromosomes. The identical hybridization patterns in individuals with or without the GSTM1 gene suggest that this locus is a component of the Mu class GST gene cluster.

Activating mutations in the ras genes are commonly found in a wide range of human tumors. We recently cloned two mammalian genes, Son of sevenless 1 (mSos1) and Son of sevenless 2 (mSos2), whose protein products appear to be important positive regulators of ras proteins. Given the proposed role of Sos proteins in ras regulation, and the frequent occurrence of activated ras alleles in tumor cells, we were interested in determining whether the Sos genes may also be activated inappropriately by DNA rearrangement in tumor cells. To investigate this possibility, we have determined the chromosomal locations of both the mouse and the human Sos1 and Sos2 genes, using a combination of genetic linkage analysis and in situ hybridization to chromosomal spreads. We find that the murine Sos1 and Sos2 genes map to chromosomes 17E and 12C3.3-D and their human counterparts to chromosomes 2p21-2p2 and 14q21, respectively. Neither the human nor the mouse Sos loci map close to known mutations or to regions showing consistent karyotypic abnormalities in tumor cells.

The human tumor necrosis factor locus (TNF locus) is located within the major histocompatibility complex between the class III genes and HLA-B. We recently characterized and studied two closely linked highly informative dinucleotide repeats (AC/GT)n (designated TNFa) and (TC/GA)k (designated TNFb) in the upstream region of the human TNF-beta (lymphotoxin) gene. We also characterized two linked (TC/GA) and (TC/GA)-like repeats located downstream of the TNF-alpha gene, designated TNFe and TNFd, respectively. Here, we combine these four markers together with a biallelic TC/GA repeat in the first intron of the TNF-beta gene (TNFc) and a biallelic NcoI RFLP (TNFn) to type 105 cell lines from the American Society of Histocompatibility and Immunogenetics Workshop and the Center for Human Polymorphism Studies reference panels of HLA typing cell lines. These 6 polymorphic markers define 35 distinct TNF haplotypes and together with the reference panel can be used for disease association and population genetics studies.

Double fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in decondensed human sperm nuclei. Biotinylated X chromosome specific (TRX) and digoxigenin-labeled Y chromosome specific (HRY) probes were simultaneously hybridized to sperm preparations from 12 normal healthy donors. After the hybridization, the probes were detected immunocytochemically, using two different and independent affinity systems. Ninety-six percent of the 12,636 sperm showed fluorescent labeling, of which 47.4% were haploid X and 46.8% were haploid Y. A frequency of 0.46% of XX-bearing sperm (0.28% disomic, 0.18% diploid) and 0.38% YY-bearing sperm (0.21% disomic, 0.17% diploid) was found. The overall proportions of X- and Y-bearing sperm in the ejaculates were 47.9% and 47.2%, respectively, which was not significantly different from the expected 50:50 ratio. In addition 0.21% of cells appeared to be haploid XY-bearing sperm, 0.62% were diploid XY-bearing cells, and 0.05% of cells were considered to be tetraploid cells. The application of double FISH to human sperm using X-chromosome and Y-chromosome probes has allowed a more accurate assessment of the sex chromosal complements in sperm than single FISH method and quinacrine staining for Y-bodies.

Fluorescence in situ hybridization (FISH) with DNA probes specific to chromosomes 17 and the X has been applied to human ejaculated sperm. After sperm nuclei were decondensed with EDTA and DTT, biotinylated alpha satellite DNA probes TR17 and TRX were separately used on preparations from thirteen healthy donors. After hybridization 96% of sperm were labelled with the TR17 probe and 48% of sperm were labelled with the TRX probe. Frequencies of 0.33% disomic 17 and 0.29% disomic X sperm were found. The frequencies of diploid sperm were assessed as 0.37% using the TR17 probe and 0.20% using the TRX probe which labelled only one half of the sperm; after correcting the result from the X-probe to 0.40% the two frequencies are very similar.

The localization of specific human ubiquitin genes has not been straightforward because of the conservation of the ubiquitin coding sequence and the number of processed pseudogenes. An congruent to 1.4-kb sequence from the 5'-flanking region of the UBC gene has been shown to be unique to that locus and free from dispersed repeat elements. The cloned 5'-flanking fragment has been used to probe Southern blots of DNA obtained from somatic cell hybrid cell lines. These data indicate that the UBC gene is located on chromosome 12. In situ hybridization with the 5'-flanking probe has refined the assignment to the broad chromosomal subband 12q24.3. These data show that the active ubiquitin genes are not clustered and are located on separate chromosomes. In addition, these studies demonstrate the utility of intron or flanking sequence probes in the specific chromosomal assignment of members of highly conserved gene families.

A probe from the 3' noncoding region of a murine type I keratin intermediate filament (IF) gene (Krt-1.14) localizes to band D of murine Chromosome 11 using in situ hybridization. This localization provides a physical confirmation of the assignment of the type I keratin genes by linkage analysis in the mouse. It also demonstrates that the Krt-1.14 genes are at a single locality in the mouse in contrast to the two locations on the short and long arms of chromosome 17 in humans.

Variability in the structure of the human tumor necrosis factor (TNF-alpha) or lymphotoxin (TNF-beta) genes may contribute to the functional polymorphism of the HLA gene complex. We have characterized an allelic restriction fragment length polymorphism (RFLP) of the TNF-beta gene by using the restriction endonuclease NcoI. Digestion of genomic DNA with NcoI and Southern blotting by using TNF-alpha gene probes show 5.4-kb and 10.5-kb hybridizing fragments. In Caucasian populations, the 10.5-kb fragment is present in 64 to 72% of haplotypes. The polymorphic NcoI site is located within the first intron of the TNF-beta gene. Additional restriction fragment variability was demonstrated by digestion with AccI; however, this restriction fragment variability was not allelic in nature. Rather, it was a consequence of variable DNA methylation at AccI sites within and upstream of the TNF-beta gene. In peripheral blood leukocytes, methylation of the TNF-beta AccI sites was greatest in neutrophils (TNF-beta nonproducers), and lowest in T lymphocytes (the major producers of TNF-beta). These results suggest strongly that variation in DNA methylation may play an important role in regulation of the expression of the TNF-beta gene.

The chromosomal location of the human ubiquitin genes has been evaluated by in situ hybridization. Because of the conservation of the ubiquitin sequence, coding-region probes cannot distinguish between specific ubiquitin genes and reveal ubiquitin sequences in a number of different chromosomal regions. The major sites of hybridization with a coding-region probe include 17p11.1-p12, 12p24.2-q24.32, and 2q21-q24, with weaker hybridization over 1p3, 1q4, 2q3, and 13q. Hybridization with a probe isolated from the UbB gene intron indicated that this gene is located within the region 17p11.1-17p12. This region showed the strongest hybridization with the coding-region probe and is presumably also the location of the duplicated UbB pseudogene.

Multiple human cytosolic glutathione transferases have been described. These enzymes are the products of multiple genes that can be classified into at least four evolutionary classes. The genes encoding each class appear to be clustered on distinct chromosomes. Over-expression of glutathione S-transferase (GST) isoenzymes has been implicated in drug resistance and, conversely, deficiency of GST isoenzymes has been implicated in susceptibility to carcinogens. Some GST genes are expressed at varying levels in different individuals, and there is a frequent deficiency of the Mu class GST1 isoenzyme in all the racial groups studied so far. This deficiency is due to a deletion of the GST 1 gene. The Alpha class genes are located on the short arm of chromosome 6 and are closely linked, with less than 2 kb separating some genes. There is evidence for the existence of several pseudogenes in this cluster. A complete Alpha class gene has 7 exons and extends over 13 kb. The 5' flanking region of the gene encoding the GST2 type 1 isoenzyme has been cloned and sequenced. This region contains a number of putative promoter and enhancer elements that are similar to those found in rat and mouse Alpha class genes.

Il-2, the gene coding for the cytokine IL2 in the mouse, has been localized by in situ hybridization to bands 3B or 3C on Chromosome 3, where it joins five other genes that also map to 4q in humans. The Il-2 probe also produces a small, secondary grain peak on Chromosome 11, in the vicinity of band 11A5, the significance of which has not yet been determined. The gene for the alpha chain of the IL2 receptor in the mouse, Il-2ra, localizes to bands 2A2 or 2A3 on Chromosome 2. This is the first assignment of a gene located on chromosome 10 in humans to Chromosome 2 in the mouse.

The cytokines, IL-3, IL-4, IL-5, and GM-CSF (encoded by murine genes Il-3, Il-4, Il-5, and Csfgm) belong to a family of secreted glycoprotein hormones that regulate the haemopoietic and immune systems. We demonstrate here using in situ hybridization that Il-3 and Il-5 are both probably located in the segment comprising band A5 and the proximal half of band B1 on mouse chromosome 11 with a possible location point in band B1 near its proximal interface with band A5. In studies reported elsewhere we have shown close physical linkage between Il-3 and Csfgm and also between Il-4 and Il-5. The in situ hybridization results therefore indicate that all four cytokine genes are clustered on chromosome 11 raising the possibility that they arose by ancient gene duplication.

We have characterized a human genomic clone that contains the 5' coding and 5' flanking sequences of the human parathyroid hormone-related protein gene (PTHrP). The 5' end of the gene contains three exons separated by two small introns of 60 and 165 bp, respectively. The coding region of the PTHrP gene exhibits significant structural homology to the human parathyroid hormone gene (PTH), including the position of at least two introns. However, there is no significant nucleotide sequence homology to the PTH gene within the intragenic region nor in the flanking genomic sequences. The PTHrP gene has been localized, by chromosomal in situ hybridization to bands p11 or p12, on human chromosome 12. Analysis of the 5'-noncoding DNA reveals a complex, putative regulatory region, with multiple potential transcription start points. Nucleotide sequence analysis shows the position of one consensus TATA sequence, at -514 bp, from the start of translation whereas the other regulatory domain is located at least 1 kb further 5' to this consensus TATA sequence. Evidence from the structure of a number of cDNA clones, as well as S1 nuclease and primer extension studies supports the hypothesis that the PTHrP gene contains at least two mRNA transcription start points that define two putative regulatory domains. The result of expression from these different promoters combined with an alternative splicing event would be to produce multiple forms of PTHrP mRNA that differ in the 5'-untranslated region. This analysis of the human PTHrP gene is the first report of a PTHrP gene for any species.

A pedigree showing the fragile site at Xq27 in a severely retarded female and in other less retarded carriers is described. Two of the four moderately retarded males with the fra(X)(q27) show macro-orchidism, and a variety of other features usually used to support the effects of the fra(X)(q27) are also inconsistent. A second fragile site at (10)(q23) is also present and in the two oldest females its frequency is not decreased, whereas the fra(x)(q27) is not detectable in these females although probably present. It is concluded that pedigrees showing mentally retarded females and probable X linkage should be included in studies of the fra(X)(q27).

Duplication of chromosome sub-bands 5p14.3 and 5p15.1 in a child resulted in mild mental retardation, apparently without other abnormalities. The duplicated region arises from recombination within a directly inserted segment following a shift within the short arm of the maternal chromosome 5 homologs.

Two mosaic sibling embryos of the Australian plague locust, Chortoicetes terminifera are reported with haploid and diploid cell lines in widely differing proportions. One small chromosome pair involved in the two cases has alternative morphology and a B-chromosome is present in one. In addition, G-banding identifies two medium-sized chromosome pairs and alternative states of a second small pair. Using these markers it is clear that both diploid cell lines are homozygous for the chromosomes of the corresponding haploid line. These embryos have thus developed by accidental parthenogenesis from haploid cells, some of which were duplicated by endomitosis after development began.

Three families are reported showing transmission of a previously described variant, which is not associated with any clinical abnormality. The variant involves additional material at the band 9p12, which shows homogeneous staining of intermediate density with GTL- and RBG-banding, and negative staining with CBG-banding. The region stains positively with Feulgen stain. In situ hybridization with total genomic human DNA, cloned alpha satellite, satellite III, and ribosomal DNA all show no hybridization to the 9p12 variant. Two members of one of the families show the largest 9p12 variant yet reported; two other carriers in this family have inherited a variant of decreased size. It is suggested that the 9p12 variants are homogeneously staining regions. Using the ISCN three-letter convention, this variant could be described as hsr(9)(p12).

A child is described with a de novo interstitial deletion of band 2p22 and a reciprocal translocation (3;7)(p21;q22). The child has mild developmental delay, coloboma of the right eye, and Hirschsprung's disease. The clinical and cytogenetic findings are described.

A partial cDNA clone of the glutathione S-transferase 3 gene (GST3) was obtained by screening a lambda gt11 human lung cDNA library with antiserum to human lung GST3. The sequence of this cDNA showed two base differences from the coding sequence of a GST3 cDNA isolated from a human placental cDNA library. Hybridization of the cloned GST3 cDNA to human chromosomes resulted in a primary peak of grains over band 11q13, a localization predicted by prior experiments. An unexpected strong secondary peak of grains was obtained over bands 12q13 and 12q14, indicating that there is a GST3-like gene on the long arm of chromosome 12 in man.

In Chortoicetes terminifera, G-banding, produced by the trypsin treatment of air-dried slides followed by Giemsa staining, leads to light staining gaps at the secondary constrictions on autosomal pair 6 and regions proximal to the centromere on the long arms of pair 4. The variable short arms of two of the three smallest pairs were usually flared and lightly stained after treatment. In contrast to the relatively minor response of the normal chromosome set to G-banding, the large supernumerary chromosomes of C. terminifera show a spectacular series of dark bands alternating with lightly stained gaps. Two G-band variants of the B-chromosome were found in a laboratory stock. These patterns of G-banding are discernable both at mitosis in adults and embryos of both sexes and at all stages of male meiosis. Some regions which are gaps after G-banding appear as dark bands after C-banding. Consequently the supernumerary chromosome is mainly darkly stained with C-banding. In addition the centromeres and some telomeres are C-banded along with narrow interstitial bands and polymorphic heterochromatic blocks.--C-banding was not always successful, the technique often yields a mixture of G- and C-banding. The disparity of banding between the normal complement and the B-chromosome implies that whatever the source of origin of the B it has undergone spectacular changes in organisation since its origin.

The glutathione S-transferases (GST) (glutathione transferase; EC 2.5.1.18) are a family of enzymes responsible for the metabolism of a broad range of xenobiotics and carcinogens. A cDNA clone containing the entire amino acid coding sequence of a human GST-2 subunit has been isolated using a lambda gt11 expression library. The complete nucleotide sequence and a partial restriction map are presented. The subunit is composed of 221 amino acids with a molecular weight of 25,425 before posttranslational modification. The deduced amino acid sequence is rich in lysine, which is consistent with the relatively high pI of GST-2. The human sequence shows considerable homology with the rat Ya and Yc GST sequences but little homology with the rat GSTp and Yb subunit sequences. Southern blots of restriction digests of human DNA indicate that there may be multiple GST-2 genes. In situ hybridization of the cloned cDNA to human chromosomes produces intense labeling only over band p12 on the short arm of chromosome 6 near the centromere. This indicates that the GST-2 gene(s) are located only at this site.

Absence of the right lateral rectus muscle and hypoplasia of the left was found in a child with congenital esotropia. He had mental and physical retardation, bilateral optic nerve hypoplasia, and many minor dysmorphic features, including brachycephaly, high forehead, poorly folded, low set ears, epicanthic folds, exaggerated Cupid's bow, long philtrum, and single palmar creases. Unusual features were a markedly ridged palate and a plantar crease which passed from the first and second interspace across the lateral border of the foot. He was found to have an unbalanced karyotype with duplication of chromosome segment 7q32----q34 (46,XY,der(2),inv?ins(2;7) (q21;q32q34)mat). The mother, maternal aunt, and sister of the proband all had a balanced rearrangement and were phenotypically normal.

The distribution of late-replicating segments along the chromosomes of five clones of W. virgo is described. Some, but not all of these segments correspond to C-bands. In general, the "autoradiographic profiles" (histograms of linear grain density along the length of chromosomes labeled with tritiated thymidine in late S-phase) show strong resemblances throughout the five clones. However, some significant differences exist, and these are particularly marked in the case of the Boulder clone, which is anomalous in many other respects. --A similar study has also been carried out on the two bisexual species of Warramaba ("P169" and "P196") that gave rise, by hybridization more than half a million years ago, to the parthenogenetic W. virgo. In the case of P169, the autoradiographic profiles of the three large chromosomes (X + A, B + 5, CD) which it has contributed to the W. virgo karyotype are extremely similar to those of the corresponding chromosomes in the virgo clones we have studied. In the case of P196 there is likewise, in most instances, a close resemblance of the autoradiographic profiles of the AB, X1 and CD chromosomes to those of the same chromosomes in the virgo clones, but that of the X1 shows no particular resemblance to the anomalous profile of the X1 in the Boulder clone, in which the X1 has undergone a structural reorganisation. The autoradiographic profile of the P196 CD chromosome does, however, show a much closer resemblance to that of the corresponding chromosome in the Boulder clone than to those of the CD196 in the other four virgo clones studied. These investigations confirm the considerable evolutionary stability of DNA replication patterns.

There is evidence for more than one alpha 1 acid glycoprotein (alpha 1 AGP) gene, and they all appear to be in close proximity. In situ hybridization of the cloned human cDNA p alpha 1AGP-2 to human chromosomes indicates that the alpha 1AGP genes are located between bands q31 and q34.1 on chromosome 9. This finding is in agreement with the previous assignment of the locus for alpha 1AGP to a linkage group with ABO and AK on chromosome 9.

The distribution of constitutive heterochromatin has been investigated in four chromosomal races of the grasshopper Caledia captiva (2n=23 male/24 female) by the C-banding technique. Each of the four races was found to have a distinctive banding pattern which is associated with the inter-racial differences in chromosomal rearrangements. The "Ancestral" race has a telocentric chromosome complements with large procentric C-bands which are structurally double on six pairs of chromosomes. The centromeres are unstained. The "General Purpose" race has a C-banding pattern very similar to that seen in other Acridine grasshoppers with the majority of its chromosomes showing a centromeric localisation of the bands. The two southern races, which show a complex polymorphism for presumed pericentric inversions on all twelve chromosomes, also show an unusually high level of interstitial and terminal C-bands. The different locations and numbers of these bands allow unambiguous identification of all the chromosome pairs within the complement. In two cases, there is good evidence to indicate that a C-band redistribution between acrocentric and metacentric chromosomes has occurred by pericentric inversion. Furthermore, C-band variation on the long arm of the metacentric X-chromosome indicates the presence of a large paracentric inversion. This double inversion system has involved over 95% of the X-chromosome. The interstitial and terminal C-bands probably have not resulted from heterochromatin movement within the complement but, more likely, have arisen by saltatory duplication of pre-existing sequences on the chromosome. A new nomenclature system for banded chromosomes is proposed which allows most kinds of chromosomal restructuring and rearrangement to be adequately enumerated.

Murine male meiotic preparations were probed by in situ hybridization to localize 10.5 kb of unique DNA which flanks the C gamma 1 heavy chain immunoglobulin gene on the 5' side. The probe, inserted into the plasmid vector pBR 322, was labelled with I125 dCTP using nick translation. After exposing autoradiographs, made by standard techniques of in situ hybridization, for 40 days, a strong signal was obtained on one bivalent of size appropriate to pair 12. The signal was in the distal position on chromosome 12 and it could be traced from spermatogonial divisions through to mature sperm. The signal was strongest in some well-spread stages of prophase of first spermatocytes, weakest in early spermatids, then surprisingly strong in sperm heads. This variation is probably due to differences in the packaging of the target sequence at the various stages of meiosis.

Three families with confirmed and one family with suspected male transmission of the fragile X are presented, with psychological and physical assessment of all available members. The psychological tests used were the Peabody Picture Vocabulary test and Block Design which measured verbal and non-verbal abilities, respectively. Physical status was assessed by recording dysmorphic features and by anthropometric measurements. This study demonstrated that there are appreciable differences in mental and physical status within sibships of daughters of male carriers, as well as recognizable physical alterations and intellectual impairment in the transmitting males. These findings contradict the concept that there are two distinct categories of fragile X carriers: phenotypically normal as opposed to affected. They suggest instead that the defect may be graded and emphasize the importance of intellectual deficits and physical alterations in defining the fragile X phenotype, both in low-penetrant males and female heterozygotes.

A young girl who had an active retinoblastoma in the left eye, and a retinoma or spontaneously regressed retinoblastoma in the right eye, was found to have a complex translocation-deletion involving chromosomes 13 and 10. Karyotypic analysis suggested that three simultaneous breaks had led to the interchange of centric and telomeric regions of chromosome 10 and 13, with loss of an interstitial acentric fragment from 13, which included subband 13q14.2. The child is intellectually retarded, and has the characteristic midface appearance associated with 13q-deletion syndrome. It is believed that this is the first report of a case of retinoblastoma and retinoma occurring in association with 13q-deletion syndrome.

A neonate with clinical features of the 11q23 deletion syndrome was apparently mosaic with the dominant cell line showing deletion of the chromosomal segment 11q23.3 to 11qter. The presence of a few lymphocytes with a normal karyotype indicates post-zygotic deletion of chromosome 11. The mother and brother of the propositus show folate-sensitive fragility at band 11q23.3. This case indicates in vivo deletion at a folate-sensitive fragile site.

Mitotic, nonmalignant Balb/c 3T3 cells exhibit endogenous, surface galactosyltransferase activity that does not require intercellular contact throughout the assay period. In this respect, mitotic 3T3 cells resemble malignant Balb/c 3T12 cells which similarly show no contact requirement for optimum transferase activity in any phase of their cell cycle. Previously, it was shown that randomly growing populations of 3T3 cells have lower galactosyltransferase activity when assayed under conditions which decreased cell contact. This led to the conclusion that these normal (3T3) and malignant (3T12) cells differed in that intercellular contact is required for optimum activity of surface galactosyltransferases on the normal cell type. The present data indicate that mitotic 3T3 cells may be capable of expressing enzyme activities exhibited at all times by malignant cells. That is, mitotic 3T3 cells and randomly growing 3T12 cells may readily catalyze galactosyltransferase reactions between enzymes and acceptors on the same cell. Interphase 3T3 cells, on the other hand, might require that enzymes glycosylate acceptors on adjacent cells. A model is proposed that suggests that changes in the spatial arrangement of surface enzymes and acceptors or variations in the fluidity of the cell membrane can account for this contact-related glycosylation.

Functional helper and suppressor hybridomas were constructed from fractionated Type II NFR/nude mouse splenocytes which resemble precursors of T cells. After carrier priming with rabbit erythrocytes (RE), splenocytes from Type II NFR/nude mice were fractionated on nylon wool columns. Cells from two distinct fractions (Pool 1 and Pool 2) were fused with BW5147 lymphoma cells. Hybrids were selected in HAT medium and subsequently cloned. Three hybridomas constructed using Pool 1 splenocytes were capable of complementing nude mouse PFC responses to the trinitrophenyl group (TNP) of TNP-RE. Four hybridomas constructed using Pool 2 splenocytes were observed to suppress anti-TNP PFC responses. These hybridomas express the T-cell surface markers TLa, Thy 1, Lyt 1, Lyt 2 in patterns which suggest that they represent expanded clones of T cells from precursors that are arrested in their development. They appear to be blocked short of development of carrier specificity in their functional phenotypes as regulatory T cells.

Complex chromosome rearrangements (CCR) involving multiple breaks in two chromosomes are rare. The detection of a four-break apparently balanced rearrangement involving chromosomes 1 and 3 in a child with developmental delay led us to reanalyse, using prometaphase banding, another complex two-chromosome rearrangement in a previously reported case (Fitzgerald 1974). The relationship between clinical abnormalities and apparently balanced rearrangements is discussed.

Chromosome results obtained from 1012 patients referred with developmental delay without known cause within the three years 1985 to 1987 are reported. G banding analysis and assessment of 70 cells for fragile X gave abnormal results in 84 cases: fragile X in 31 patients and other abnormalities in 53 patients. A further 16 sibs expressing the fragile X were detected in family studies originating from the 31 index cases. This yield justifies continuation of procedures which detect both fragile X and subtle chromosomal abnormalities in these patients.

Electrophoretic studies of the genetic variation of the A subunit of coagulation factor XIII (F13A) have shown that the A subunits expressed in placenta and in the circulation are the products of the same gene locus. In situ hybridization studies with a cDNA fragment encoding the amino terminal region of the A subunit have localized the gene to bands p24----p25 on chromosome 6. This confirms previous studies that showed linkage between F13A and the major histocompatibility complex.